U.S. patent application number 10/478056 was filed with the patent office on 2004-10-28 for anti-trail-r antibodies.
Invention is credited to Kataoka, Shiro, Mori, Eiji.
Application Number | 20040214235 10/478056 |
Document ID | / |
Family ID | 27346753 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214235 |
Kind Code |
A1 |
Mori, Eiji ; et al. |
October 28, 2004 |
Anti-trail-r antibodies
Abstract
Anti-TRAIL-R1 and R2 antibodies or functional fragments thereof,
having at least one property selected from the following (a) to (c)
of: (a) having activity to induce apoptosis in carcinoma cells
expressing TRAIL-R1 and/or TRAIL-R2; (b) not having effect on
normal human cells expressing TRAIL-R1 and/or TRAIL-R2; and (c) not
inducing human hepatocyte toxicity, and an anti-TRAIL-R1 and R2
antibodies or functional fragments thereof, having the following
properties: having activity to induce apoptosis in carcinoma cells
independently of exogenous factors and as a monomer of an
antibody.
Inventors: |
Mori, Eiji; (Gunma, JP)
; Kataoka, Shiro; (Gunma, JP) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Family ID: |
27346753 |
Appl. No.: |
10/478056 |
Filed: |
June 7, 2004 |
PCT Filed: |
May 17, 2002 |
PCT NO: |
PCT/JP02/04816 |
Current U.S.
Class: |
435/7.2 ;
530/388.22 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/00 20180101; C07K 2317/73 20130101; A61P 43/00 20180101;
A61P 35/02 20180101; C07K 16/2878 20130101 |
Class at
Publication: |
435/007.2 ;
530/388.22 |
International
Class: |
G01N 033/53; G01N
033/567; C07K 016/28 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 2001 |
JP |
2001-150213 |
Aug 9, 2001 |
JP |
2001-243040 |
Oct 11, 2001 |
JP |
2001-314489 |
Claims
1. An antibody or a functional fragment thereof, binding to
TRAIL-R1 and/or Trail-R2.
2. The antibody or the functional fragment thereof of claim 1,
having at least one property selected from the following (a) to (c)
of: (a) having activity to induce apoptosis in carcinoma cells
expressing TRAIL-R1 and/or TRAIL-R2; (b) not having effect on
normal human cells expressing TRAIL-R1 and/or TRAIL-R2; and (c) not
inducing human hepatocyte toxicity.
3. An antibody or a functional fragment thereof, having all the
following properties (a) to (c) of: (a) having activity to induce
apoptosis in carcinoma cells expressing TRAIL-R1 and/or TRAIL-R2;
(b) not having effect on normal human cells expressing TRAIL-R1
and/or TRAIL-R2; and (c) not inducing human hepatocyte
toxicity.
4. The antibody or the functional fragment thereof of claim 2 or 3,
which binds to TRAIL-R2, but does not bind to TRAIL-R1.
5. The antibody or the functional fragment thereof of claim 2 or 3,
which binds to both TRAIL-R2 and TRAIL-R1.
6. The antibody or the functional fragment thereof of any one of
claims 1 to 5, which is a monoclonal antibody produced by a
mouse-mouse hybridoma.
7. The antibody or the functional fragment thereof of any one of
claims 1 to 6, which is a human antibody.
8. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 0.01 .mu.g/ml or more for
human hepatocytes when the number of cells is 7.5.times.10.sup.4
and the reaction time is 24 hours.
9. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 0.1 .mu.g/ml or more for
human hepatocytes when the number of cells is 7.5.times.10.sup.4
and the reaction time is 24 hours.
10. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 2 to 10 .mu.g/ml for human
hepatocytes when the number of cells is 7.5.times.10.sup.4 and the
reaction time is 24 hours.
11. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 10 .mu.g/ml or more for
human hepatocytes when the number of cells is 7.5.times.10.sup.4
and the reaction time is 24 hours.
12. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 10 to 100 .mu.g/ml for human
hepatocytes when the number of cells is 7.5.times.10.sup.4 and the
reaction time is 24 hours.
13. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 100 .mu.g/ml or more for
human hepatocytes when the number of cells is 7.5.times.10.sup.4
and the reaction time is 24 hours.
14. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 100 .mu.g/ml or less for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
15. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 10 .mu.g/ml or less for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
16. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 0.7 .mu.g/ml or less for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
17. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 0.02 to 0.11 .mu.g/ml for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
18. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 0.02 .mu.g/ml or less for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
19. The antibody or the functional fragment thereof of any one of
claims 1 to 7, having an LD50 value of 2 to 100 .mu.g/ml for human
hepatocytes when the number of cells is 7.5.times.10.sup.4 and the
reaction time is 24 hours, and having an LD50 value of 0.02 to 0.11
.mu.g/ml for carcinoma cells when the number of cells is
2.5.times.10.sup.3 and the reaction time is 48 hours.
20. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 2 times or more greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
21. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 10 times or more greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
22. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 50 times or more greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
23. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 50 to 100 times greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
24. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 100 times or more greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
25. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 100 to 1000 times greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
26. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 250 to 1000 times greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
27. The antibody or the functional fragment thereof of any one of
claims 1 to 7, wherein the LD50 value for human hepatocytes when
the number of cells is 7.5.times.10.sup.4 and the reaction time is
24 hours is 1000 times or more greater than the LD50 value for
carcinoma cells when the number of cells is 2.5.times.10.sup.3 and
the reaction time is 48 hours.
28. The antibody or the functional fragment thereof of any one of
claims 8 to 27, wherein a reaction volume is between 110 and 120
.mu.l.
29. The antibody or the functional fragment thereof of any one of
claims 2, 3 and 14 to 28, wherein the carcinoma cells are Colo205
cells.
30. The antibody or the functional fragment thereof of claim 2 or
3, wherein the carcinoma cells are Colo205 cells, U251 cells or
Jurkat cells.
31. The antibody or the functional fragment thereof of any one of
claims 1 to 30, which can suppress tumor growth or regress
tumors.
32. The antibody or the functional fragment thereof of claim 31,
wherein the tumor is at least one tumor selected from the group
consisting of colon cancer, colorectal cancer, lung cancer, breast
cancer, brain tumor, malignant melanoma, renal cell carcinoma,
bladder cancer, leukemia, lymphomas, T cell lymphomas, multiple
myeloma, gastric cancer, pancreas cancer, cervical cancer,
endometrial carcinoma, ovarian cancer, esophageal cancer, liver
cancer, head and neck squamous cell carcinoma, cutaneous cancer,
urinary tract carcinoma, prostate cancer, choriocarcinoma,
pharyngeal cancer, laryngeal cancer, thecomatosis, androblastoma,
endometrium hyperplasy, endometriosis, embryoma, fibrosarcoma,
Kaposi's sarcoma, hemangioma, cavernous hemangioma, angioblastoma,
retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma,
medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma,
hamartoblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid
sarcoma, Wilms tumor and the like.
33. The antibody or the functional fragment thereof of claim 31,
wherein the tumor is derived from Colo205 cells transplanted in a
nude mouse.
34. The antibody or the functional fragment thereof of any one of
claims 31 to 33, wherein a period during which tumor growth can be
suppressed or tumor regression can be achieved is at least 9
days.
35. The antibody or the functional fragment thereof of any one of
claims 31 to 34, wherein the dose of the antibody or the functional
fragment thereof is 100 .mu.g/body or 5 mg/kg.
36. The antibody or the functional fragment thereof of any one of
claims 31 to 34, wherein the dose of the antibody or the functional
fragment thereof is 20 .mu.g/body or 1 mg/kg.
37. The antibody or the functional fragment thereof of any one of
claims 31 to 34, wherein the dose of the antibody or the functional
fragment thereof is 4 .mu.g/body or 200kg/kg.
38. The antibody or the functional fragment thereof of any one of
claims 31 to 34, wherein the dose of the antibody or the functional
fragment thereof is 1 .mu.g/body or 50 .mu.g/kg.
39. The antibody or the functional fragment thereof of any one of
claims 1 to 38, which is an immunoglobulin G antibody.
40. An antibody or a functional fragment thereof, which can induce
an average of 14% or more tumor reduction by 4 days after the
initial administration, when administered at a concentration of 20
.mu.g/mouse to a 4- to 6-week-old tumor-bearing mouse having a 100
mm.sup.3 tumor.
41. The antibody or the functional fragment thereof of claim 40,
which can maintain an average of 14% or more tumor reduction for at
least 7 days.
42. The antibody or the functional fragment thereof of claim 40,
which can induce an average of 65% or more tumor reduction by 4
days after the initial administration, when administered at a
concentration of 20 .mu.g/mouse to a 4- to 6-week-old tumor-bearing
mouse having a 100 mm.sup.3 tumor.
43. The antibody or the functional fragment thereof of claim 40,
which can induce an average of 80% or more tumor reduction by 7
days after the initial administration, when administered at a
concentration of 20 .mu.g/mouse to a 4- to 6-week-old tumor-bearing
mouse having a 100 mm.sup.3 tumor.
44. The antibody or the functional fragment thereof of claim 43,
which can maintain an average of 80% or more tumor reduction for at
least 4 days.
45. The antibody or the functional fragment thereof of claim 40,
which can induce an average of 45% or more tumor reduction by 3
days after the initial administration, when administered at a
concentration of 25 .mu.g/mouse to a 12-week-old tumor-bearing
mouse having a 100 mm.sup.3 tumor.
46. The antibody or the functional fragment thereof of claim 45,
which can induce an average of 65% or more tumor reduction by 5
days after the initial administration, when administered at a
concentration of 25 .mu.g/mouse to a 12-week-old tumor-bearing
mouse having a 100 mm.sup.3 tumor.
47. The antibody or the functional fragment thereof of claim 46,
which can maintain an average of 65% or more tumor reduction for at
least 27 days.
48. The antibody or the functional fragment thereof of claim 40,
which can induce an average of 39% or more tumor reduction by 4
days after the initial administration, when administered at a
concentration of 20 .mu.g/mouse to a 4- to 6-week-old tumor-bearing
mouse having a 300 mm.sup.3 tumor.
49. The antibody or the functional fragment thereof of claim 48,
which can maintain an average of 39% or more tumor reduction for at
least 14 days.
50. The antibody or the functional fragment thereof of claim 40,
which is a 0304 antibody.
51. The antibody or the functional fragment thereof of claim 40,
which is an E-11-13 antibody.
52. An antibody or a functional fragment thereof binding to
TRAIL-R1 and/or TRAIL-R2, which is produced by a hybridoma E-11-13,
H-48-2, L-30-10, N-1 8-12, W-40-5, X-14-4, X-51-12, F-4-8, G-3-10,
0304 or KMTR1.
53. An antibody or a functional fragment thereof binding to
TRAIL-R1 and/or TRAIL-R2, which is produced by a hybridoma H-48-2
with the accession number of FERM BP-7599, a hybridoma E-11-13 with
the accession number of FERM BP-7698 or FERM BP-7770, a hybridoma
F-4-8 with the accession number of FERM BP-7699 or FERM BP-7768, a
hybridoma L-30-10 with the accession number of FERM BP-7700 or FERM
BP-7769, a hybridoma 0304 with the accession number of FERM
BP-8037, or a hybridoma KMTR1 with the accession number of FERM
BP-8038.
54. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma E-11-13, which are respectively represented by SEQ ID
NOS: 17 and 19; a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma L-30-10,
which are respectively represented by SEQ ID NOS: 21 and 23; a
heavy chain variable region and a light chain variable region of
the antibody produced by a hybridoma H-48-2, which are respectively
represented by SEQ ID NOS: 25 and 27; a heavy chain variable region
and a light chain variable region of the antibody produced by a
hybridoma 0304, which are respectively represented by SEQ ID NOS:
29 and 31; or a heavy chain variable region and a light chain
variable region of the antibody produced by a hybridoma KMTR1,
which are respectively represented by SEQ ID NOS: 33 and 35.
55. An antibody or a functional fragment thereof, having amino acid
sequences of the mature portions of a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma E-11-13, which are respectively
represented by SEQ ID NOS: 16 and 18; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma L-30-10, which are respectively
represented by SEQ ID NOS: 20 and 22; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma H-48-2, which are respectively
represented by SEQ ID NOS: 24 and 26; a heavy chain variable region
and a light chain variable region that are encoded by nucleic acid
sequences isolated from a hybridoma 0304, which are respectively
represented by SEQ ID NOS: 28 and 30; or a heavy chain variable
region and a light chain variable region that are encoded by
nucleic acid sequences isolated from a hybridoma KMTR1, which are
respectively represented by SEQ ID NOS: 32 and 34.
56. A hybridoma producing monoclonal antibodies that bind to
TRAIL-R2, which is selected from the group consisting of E-11-13,
H-48-2, L-30-10, N-1 8-12, W-40-5, X-14-4, X-51-12, F-4-8, G-3-10,
0304 and KMTR1.
57. A hybridoma producing monoclonal antibodies that bind to
TRAIL-R2, which is selected from a hybridoma H-48-2 with the
accession number of FERM BP-7599, a hybridoma E-11-13 with the
accession number of FERM BP-7698 or FERM BP-7770, a hybridoma F-4-8
with the accession number of FERM BP-7699 or FERM BP-7768, a
hybridoma L-30-10 with the accession number of FERM BP-7700 or FERM
BP-7769, a hybridoma 0304 with the accession number of FERM BP-8037
and a hybridoma KMTR1 with the accession number of FERM
BP-8038.
58. A method for producing anti-TRAIL-R2 monoclonal antibodies,
comprising culturing the hybridoma of claim 56 or 57, and
collecting the antibodies binding to TRAIL-R2 from the obtained
culture product.
59. A method for producing anti-TRAIL-R2 monoclonal antibodies,
comprising isolating a gene encoding an anti-TRAIL-R2 monoclonal
antibody from the hybridoma of claim 56 or 57, constructing an
expression vector having the gene, introducing the expression
vector into a host to express the monoclonal antibody, and
collecting anti-TRAIL-R2 monoclonal antibodies from the obtained
host, or the culture supernatant or the secretion of the host.
60. The production method of claim 59, wherein the host is any host
selected from the group consisting of Escherichia coli, yeast
cells, insect cells, mammalian cells and plant cells, and
mammals.
61. A prophylactic or therapeutic agent against tumors, comprising
as an active ingredient the antibody or the functional fragment
thereof of any one of claims 1 to 55.
62. The prophylactic or therapeutic agent of claim 61, wherein the
tumor is any one tumor selected from the group consisting of colon
cancer, colorectal cancer, lung cancer, breast cancer, brain tumor,
malignant melanoma, renal cell carcinoma, bladder cancer, leukemia,
lymphomas, T cell lymphomas, multiple myeloma, gastric cancer,
pancreas cancer, cervical cancer, endometrial carcinoma, ovarian
cancer, esophageal cancer, liver cancer, head and neck squamous
cell carcinoma, cutaneous cancer, urinary tract carcinoma, prostate
cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer,
thecomatosis, androblastoma, endometrium hyperplasy, endometriosis,
embryoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous
hemangioma, angioblastoma, retinoblastoma, astrocytoma,
neurofibroma, oligodendroglioma, medulloblastoma,
ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma,
osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma, Wilms tumor
and the like.
63. An antibody or a functional fragment thereof of any one of
claims 1 to 49, which binds to TRAIL-R and induces apoptosis in
carcinoma cells expressing TRAIL-R as a monomer independently of
exogenous factors.
64. An antibody or a functional fragment thereof of any one of
claims 1 to 49, which binds to TRAIL-R and induces apoptosis in
carcinoma cells expressing TRAIL-R as a monomer independently of
exogenous factors, and the survival rate of carcinoma cells in the
following test using the said antibody or functional fragment
thereof is 80% or less, (1) Preparing Colo205 cells (ATCC
No.CCL-222) which were colon carcinoma cells, at a concentration of
1.0.times.10.sup.5/ml in RPMI-1640 medium containing 10% FCS,
adding the cells to each well of a 96-well flat-bottomed plate at
100 .mu.l/well and culturing at 37.degree. C. under 5.0% carbon
dioxide gas for 24 hours, (2) Adding to each well of (1) an
antibody or a functional fragment thereof which is bound to TRAIL-R
dissolved in RPMI-1640 medium containing 10% FCS such that a
concentration of the antibody or the functional fragment thereof
becomes 1000 ng/ml when it is added to each well at 10 .mu.l/well,
culturing each well at 37.degree. C. under 5.0% carbon dioxide gas
for 48 hours, washing each well once with PBS and adding a fresh
RPMI-1640 medium containing 10% FCS at 100 .mu.l/well, (3) Adding
20 .mu.l of MTS reagent (Cell Titer 960 AQ.sub.UEOUS
Non-Radioactive Cell Proliferation Assay: Promega) to each well of
(2) and culturing at 37.degree. C. under 5.0% carbon dioxide gas
for 2 hours, and (4) Measuring absorbance of each well of (3) at a
wavelength of 490 nm (with a reference wavelength of 630 nm) using
a microplate reader and calculating the survival rate of the cells
using the reducibility of the mitochondria as an indicator, wherein
the survival rate of the cells is calculates using the-following
formula, Survival rate (%)=100.times.(a-b)/(c-b) (wherein "a"
represents the measured value of a well tested, "b" represents the
measured value of a carcinoma cell-free well, and "c" represents
(i) the measured value of a well containing carcinoma cells and a
control antibody which is not bound to carcinoma cells and has the
same subclass with the antibody or the functional fragment thereof
bound to TRAIL-R when the antibody or the functional fragment
thereof has a constant region, or (ii) the measured value of a well
containing carcinoma cells and a control antibody which is not
bound to the carcinoma cells and does not have a constant region
when the antibody or the functional fragment thereof does not have
a constant region).
65. An antibody or a functional fragment thereof of claim 64,
wherein the survival rate is 60% or less.
66. An antibody or a functional fragment thereof of claim 64,
wherein the survival rate is 40% or less.
67. An antibody or a functional fragment thereof of claim 64,
wherein the survival rate is 20% or less.
68. An antibody or a functional fragment thereof of claim 64,
wherein the survival rate is 10% or less.
69. An antibody of any one of claims 1 to 49, which binds to
TRAIL-R and induces apoptosis in carcinoma cells expressing TRAIL-R
as a monomer independently of exogenous factors, and the survival
rate of carcinoma cells in the following test using the said
antibody is 80% or less, (1) Preparing Colo205 cells (ATCC
No.CCL-222) which were colon carcinoma cells, at a concentration of
1.0.times.10.sup.5/ml in RPMI-1640 medium containing 10% FCS,
adding the cells to each well of a 96-well flat-bottomed plate at
100 .mu.l/well and culturing at 37.degree. C. under 5.0% carbon
dioxide gas for 24 hours, (2) Adding to each well of (1) an
antibody which is bound to TRAIL-R dissolved in RPMI-1640 medium
containing 10% FCS such that a concentration of the antibody
becomes 1000 ng/ml when it is added to each well at 10 .mu.l/well,
culturing each well at 37.degree. C. under 5.0% carbon dioxide gas
for 48 hours, washing each well once with PBS and adding a fresh
RPMI-1640 medium containing 10% FCS at 100.mu.l/well, (3) Adding 20
.mu.l of MTS reagent (Cell Titer 969 AQ.sub.UEOUS Non-Radioactive
Cell Proliferation Assay: Promega) to each well of (2) and
culturing at 37.degree. C. under 5.0% carbon dioxide gas for 2
hours, and (4) Measuring absorbance of each well of (3) at a
wavelength of 490 nm (with a reference wavelength of 630 nm) using
a microplate reader and calculating the survival rate of the cells
using the reducibility of the mitochondria as an indicator, wherein
the survival rate of the cells is calculates using the following
formula, Survival rate (%)=100.times.(a-b)/(c-b) (wherein "a"
represents the measured value of a well tested, "b" represents the
measured value of a carcinoma cell-free well, and "c" represents
the measured value of a well containing carcinoma cells and a
control antibody which has the same subclass with the antibody
bound to TRAIL-R and is not bound to the carcinoma cells).
70. An antibody of claim 69, wherein the survival rate is 60% or
less.
71. An antibody of claim 69, wherein the survival rate is 40% or
less.
72. An antibody of claim 69, wherein the survival rate is 20% or
less.
73. An antibody of claim 69, wherein the survival rate is 10% or
less.
74. An antibody of any one of claims 1 to 49, which binds to
TRAIL-R and induces apoptosis in carcinoma cells expressing TRAIL-R
as a monomer independently of exogenous factors, and the survival
rate of carcinoma cells in the following test using the said
antibody is 80% or less, (1) Preparing Colo205 cells (ATCC
No.CCL-222) which were colon carcinoma cells, at a concentration of
5.times.10.sup.4/ml in RPMI-1640 medium containing 10% FCS, adding
the cells to each well of a 96-well flat-bottomed plate at 100
.mu.l/well and culturing at 37.degree. C. under 5.0% carbon dioxide
gas for 24 hours, (2) Adding to each well of (1) an antibody which
is bound to TRAIL-R dissolved in RPMI-1640 medium containing 10%
FCS such that a concentration of the antibody becomes 1000 ng/ml
when it is added to each well at 10 .mu.l/well, culturing at
37.degree. C. under 5.0% carbon dioxide gas for 1 hour, adding a
control antibody which has the same subclass with the antibody
bound to TRAIL-R and is not bound to carcinoma cells such that a
concentration is 100 .mu.g/ml, adding goat anti-human IgG
(7)-specific polyclonal antibodies at a final concentration of 10
.mu.g/ml, culturing each well at 37.degree. C. under 5.0% carbon
dioxide gas for 2 days, washing each well once with PBS and adding
a fresh RPMI-1640 medium containing 10% FCS at 100 .mu.l/well, (3)
Adding 20 .mu.l of MTS reagent (Cell Titer 96.RTM. AQ.sub.UEOUS
Non-Radioactive Cell Proliferation Assay: Promega) to each well of
(2) and culturing at 37.degree. C. under 5.0% carbon dioxide gas
for 2 hours, and (4) Measuring absorbance of each well of (3) at a
wavelength of 490 nm (with a reference wavelength of 630 nm) using
a microplate reader and calculating the survival rate of the cells
using the reducibility of the mitochondria as an indicator, wherein
the survival rate of the cells is calculates using the following
formula, Survival rate (%)=100.times.(a-b)/(c-b) (wherein "a"
represents the measured value of a well tested, "b" represents the
measured value of a carcinoma cell-free well, and "c" represents
the measured value of a well containing carcinoma cells and a
control antibody which has the same subclass with the antibody
bound to TRAIL-R and is not bound to the carcinoma cells).
75. An antibody of claim 74, wherein the survival rate is 60% or
less.
76. An antibody of claim 74, wherein the survival rate is 40% or
less.
77. An antibody of claim 74, wherein the survival rate is 20% or
less.
78. An antibody of claim 74, wherein the survival rate is 10% or
less.
79. An antibody of any one of claims 1 to 49, which binds to
TRAIL-R and induces apoptosis in carcinoma cells expressing TRAIL-R
as a monomer independently of exogenous factors, and the survival
rate of carcinoma cells in the following test using the said
antibody is 80% or less, (1) Preparing Colo205 cells (ATCC
No.CCL-222) which were colon carcinoma cells, at a concentration of
5.times.10.sup.4/ml in RPMI-1640 medium containing 10% FCS, adding
the cells to each well of a 96-well flat-bottomed plate at 100
.mu.l/well and culturing at 37.degree. C. under 5.0% carbon dioxide
gas for 24 hours, (2) Adding to each well of (1) an antibody which
is bound to TRAIL-R dissolved in RPMI-1640 medium containing 10%
FCS such that a concentration of the antibody becomes 1000 ng/ml
when it is added to each well at 10 .mu.l/well, culturing at
37.degree. C. under 5.0% carbon dioxide gas for 1 hour, adding a
control antibody which has the same subclass with the antibody
bound to TRAIL-R and is not bound to carcinoma cell such that a
concentration is 3 .mu.g/ml, adding goat anti-human IgG
(.gamma.)-specific polyclonal antibodies at a final concentration
of 10 g/ml, culturing each well at 37.degree. C. under 5.0% carbon
dioxide gas for 2 days, washing each well once with PBS and adding
a fresh RPMI-1640 medium containing 10% FCS at 100 .mu.l/well, (3)
Adding 20 .mu.l of MTS reagent (Cell Titer 960 AQ.sub.UEOUS
Non-Radioactive Cell Proliferation Assay: Promega) to each well of
(2) and culturing at 37.degree. C. under 5.0% carbon dioxide gas
for 2 hours, and (4) Measuring absorbance of each well of (3) at a
wavelength of 490 nm (with a reference wavelength of 630 nm) using
a microplate reader and calculating the survival rate of the cells
using the reducibility of the mitochondria as an indicator, wherein
the survival rate of the cells is calculates using the following
formula, Survival rate (%)=100.times.(a-b)/(c-b) (wherein "a"
represents the measured value of a well tested, "b" represents the
measured value of a carcinoma cell-free well, and "c" represents
the measured value of a well containing carcinoma cells and a
control antibody which has the same subclass with the antibody
bound to TRAIL-R and is not bound to the carcinoma cells).
80. An antibody of claim 79, wherein the survival rate is 60% or
less.
81. An antibody of claim 79, wherein the survival rate is 40% or
less.
82. An antibody of claim 79, wherein the survival rate is 20% or
less.
83. An antibody of claim 79, wherein the survival rate is 10% or
less.
84. An antibody or a functional fragment thereof of any one of
claims 1 to 49, which binds to TRAIL-R and induces apoptosis in
carcinoma cells expressing TRAIL-R as a monomer independently of
exogenous factors, and the survival rate of carcinoma cells on
condition that (1)1.0.times.10.sup.5/ml of carcinoma cells and
(2)1000ng/ml of the antibody or the functional fragment thereof are
cultured at 37.degree. C. under 5.0% carbon dioxide gas for 48
hours is 80% or less,
85. An antibody or a functional fragment thereof of claim 84,
wherein the survival rate is 60% or less.
86. An antibody or a functional fragment thereof of claim 84,
wherein the survival rate is 40% or less.
87. An antibody or a functional fragment thereof of claim 84,
wherein the survival rate is 20% or less.
88. An antibody or a functional fragment thereof of claim 84,
wherein the survival rate is 10% or less.
89. An antibody or a functional fragment thereof of any one of
claims 1 to 49, which binds to TRAIL-R and induces apoptosis in
carcinoma cells expressing TRAIL-R as a monomer independently of
exogenous factors, and the survival rate of carcinoma cells on
condition that (1)5.times.10.sup.4/ml of carcinoma cells,
(2)1000ng/ml of the antibody, (3)100 .mu.g/ml of a control antibody
or a functional fragment thereof which has the same subclass with
the antibody or the functional fragment thereof bound to TRAIL-R
and is not bound to carcinoma cells and (4)an antibody which binds
to both the antibody or the functional fragment thereof bound to
TRAIL-R and the control antibody are cultured at 37.degree. C.
under 5.0% carbon dioxide gas for 48 hours is 80% or less.
90. An antibody or a functional fragment thereof of claim 89,
wherein the survival rate is 60% or less.
91. An antibody or a functional fragment thereof of claim 89,
wherein the survival rate is 40% or less.
92. An antibody or a functional fragment thereof of claim 89,
wherein the survival rate is 20% or less.
93. An antibody or a functional fragment thereof of claim 89,
wherein the survival rate is 10% or less.
94. An antibody or a functional fragment thereof of any one of
claims 1 to 49, which binds to TRAIL-R and induces apoptosis in
carcinoma cells expressing TRAIL-R as a monomer independently of
exogenous factors, and the survival rate of carcinoma cells on
condition that (1)5.times.10.sup.4/ml of carcinoma cells, (2)1000
ng/ml of the antibody, (3)3 .mu.g/ml of a control antibody or a
functional fragment thereof which has the same subclass with the
antibody or the functional fragment thereof bound to TRAIL-R and is
not bound to carcinoma cells and (4)an antibody which binds to both
the antibody or the functional fragment thereof bound to TRAIL-R
and the control antibody are cultured at 37.degree. C. under 5.0%
carbon dioxide gas for 48 hours is 80% or less.
95. An antibody or a functional fragment thereof of claim 94,
wherein the survival rate is 60% or less.
96. An antibody or a functional fragment thereof of claim 94,
wherein the survival rate is 40% or less.
97. An antibody or a functional fragment thereof of claim 94,
wherein the survival rate is 20% or less.
98 An antibody or a functional fragment thereof of claim 94,
wherein the survival rate is 10% or less.
99. An antibody or a functional fragment thereof of any one of
claims 84 to 98, wherein the carcinoma cell is Colo205.
100. An antibody or a functional fragment thereof which is bound to
TRAIL-R of any one of claims 1 to 49, the activity to induce
apoptosis of which antibody or a functional fragment thereof on
carcinoma cells expressing TRAIL-R does not substantially change
depending on the presence or absence of an antibody which is bound
to a constant region of the said antibody which is bound to
TRAIL-R.
101. An antibody or a functional fragment thereof which is bound to
TRAIL-R of any one of claims 1 to 49, wherein the survival rate of
carcinoma cells expressing TRAIL-R does not substantially change
depending on the presence or absence of an antibody which is bound
to a constant region of the said antibody which is bound to
TRAIL-R.
102. A therapeutic composition, comprising as an active ingredient
the antibody or the functional fragment thereof of any one of
claims 63 to 101.
103. A prophylactic or therapeutic agent against tumors, comprising
as an active ingredient the antibody or the functional fragment
thereof of any one of claims 63 to 101.
104. A prophylactic or therapeutic agent against tumors of claim
103, wherein the tumor is any one tumor selected from the group
consisting of colon cancer, colorectal cancer, lung cancer, breast
cancer, brain tumor, malignant melanoma, renal cell carcinoma,
bladder cancer, leukemia, lymphomas, T cell lymphomas, multiple
myeloma, gastric cancer, pancreas cancer, cervical cancer,
endometrial carcinoma, ovarian cancer, esophageal cancer, liver
cancer, head and neck squamous cell carcinoma, cutaneous cancer,
urinary tract carcinoma, prostate cancer, choriocarcinoma,
pharyngeal cancer, laryngeal cancer, thecomatosis, androblastoma,
endometrium hyperplasy, endometriosis, embryoma, fibrosarcoma,
Kaposi's sarcoma, hemangioma, cavernous hemangioma, angioblastoma,
retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma,
medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma,
hamartoblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid
sarcoma, Wilms tumor and the like.
105. A method of preventing or treating tumors, comprising
administering the antibody or the functional fragment thereof of
any one of claims 63 to 101 to a patient.
106. A method of preventing or treating tumors of claim 105,
wherein the tumor is any one tumor selected from the group
consisting of colon cancer, colorectal cancer, lung cancer, breast
cancer, brain tumor, malignant melanoma, renal cell carcinoma,
bladder cancer, leukemia, lymphomas, T cell lymphomas, multiple
myeloma, gastric cancer, pancreas cancer, cervical cancer,
endometrial carcinoma, ovarian cancer, esophageal cancer, liver
cancer, head and neck squamous cell carcinoma, cutaneous cancer,
urinary tract carcinoma, prostate cancer, choriocarcinoma,
pharyngeal cancer, laryngeal cancer, thecomatosis, androblastoma,
endometrium hyperplasy, endometriosis, embryoma, fibrosarcoma,
Kaposi's sarcoma, hemangioma, cavernous hemangioma, angioblastoma,
retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma,
medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma,
hamartoblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid
sarcoma, Wilms tumor and the like.
107. A method of inducing apoptosis in carcinoma cells expressing
TRAIL-R independently of exogenous factors, which comprises
contacting an antibody or a functional fragment threreof of any one
of claims 63 to 101 with carcinoma cells expressing TRAIL-R.
108. A method of producing an antibody or a functional fragment
thereof of any one of claims 63 to 101, which comprisies, (i) a
step of immunizing an animal with TRAIL-R or a fragment thereof
having the antigenicity, cells expressing the TRAIL-R or a fragment
thereof having the antigenicity, or a DNA containing the gene
encoding all or a part of the extracellular domain of TRAIL-R, (ii)
a step of obtaining antibodies from the animal, (iii) a step of
evaluating the activity of the antibodies to induce apoptosis in
carcinoma cells expressing TRAIL-R independently of exogenous
factors, (iv) a step of separating a monomer antibody from the
antibody, (v) a step of evaluating the activity to induce apoptosis
of the said monomer antibody, and (vi) a step of selecting a
monomer antibody having the activity to induce apoptosis.
Description
[0001] This a Continuation-In-Part Application of PCT/JP02/04816
filed May 17, 2002, and is incorporated herein in its entirety by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to an anti-TRAIL receptor
(TRAIL-R) antibody recognizing a TRAIL receptor 1 (TRAIL-R1) or a
TRAIL receptor 2 (TRAIL-R2), which are cell membrane molecules
involved in apoptosis.
[0003] Furthermore, the present invention relates to a prophylactic
or therapeutic agent, which contains anti-TRAIL-R antibody as an
active ingredient and is used against diseases caused by cells
expressing TRAIL-R, and in particular relates to a therapeutic
agent used against malignant tumors.
[0004] Furthermore, the present invention relates to a prophylactic
or therapeutic agent, which contains anti-TRAIL-R antibody having
the activity to induce apoptosis as a monomer independently of
exogenous factors as an active ingredient and is used against
diseases caused by cells expressing TRAIL-R, and in particular
relates to a therapeutic agent used against malignant tumors.
BACKGROUND OF THE INVENTION
[0005] In the living body, physiological cell death caused by
normal cell alternation is referred to as apoptosis, and is
distinguished from necrosis, which is pathological cell death [see
Kerr, et al. (1972) Br. J. Cancer 26, 239]. Apoptosis is the
phenomenon generally observed in the process of, for example,
embryogenesis and the selection of lymphocytes (T cells and B
cells) [see Itoh, S., et al. (1991) Cell 66, 233-243]. It is
thought that when cells which should originally be eliminated by
apoptosis are not removed, this may cause cancer, lupus, herpes
virus infection, hepatitis and other problems. Moreover, when cells
that originally should survive are eliminated by apoptosis, this
can cause diseases and pathological conditions such as AIDS,
Alzheimer disease, Parkinson disease, amyotrophic lateral
sclerosis, multiple sclerosis, retinitis pigmentosa, aplastic
anemia, myocardial infarction, cerebral apoplexy or toxic
substances-induced hepatopathy [see Kataoka, S., et al. (1996) The
Oncologist 1, 399-401; see Mundt, B., et al.(2003) FASEB 17, 94;
see Ichikawa, K., et al. (2003) J. Immunol. 171, 1061-1069].
[0006] During apoptosis, characteristic phenomena such as curved
cell surfaces, condensation of nuclear chromatin, fragmentation of
chromosomal DNA, and loss of mitochondrial function are observed.
Various intrinsic and extrinsic signals are thought to cause these
cellular changes. As intrinsic signals, it has been reported that
oncogenes such as myc and bcl-2 and tumor suppressor genes such as
p53 are involved in apoptosis induction [see KATAOKA et al., (1993)
JIKKEN IGAKU 11, 17, 2324-2328]. As extrinsic signals, it is known
that chemotherapy drugs, radiation or the like induces apoptosis
[see KATAOKA et al., (1994) SAISHIN IGAKU 49, 6, 1152-1157].
[0007] As molecules involved in such apoptosis, molecules belonging
to tumor necrosis factor family (TNF family) such as tumor necrosis
factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta.
(TNF-.beta.) and Fas ligand have been identified. It has been
reported that these molecules are bound to specific receptors on
the surface of cells to transduce a signal to cells and induce
apoptosis in various cells. TNF-.alpha. and TNF-.beta. have been
reported to induce apoptosis in carcinoma cells [see Schmid et al.,
(1986) Proc. Natl. Acad. Sci. 83, 1881; see Dealtry et al., (1987)
Eur. J. Immunol. 17, 689]. Since mice having mutant Fas or Fas
ligands develop the conditions of autoimmune disease, it has been
strongly suggested that the Fas ligands have a function of
eliminating self-reactive lymphocytes by apoptosis in the periphery
[see Krammer, et al., (1994) Curr. Op. Immunol. 6, 279-289; see
Nagata, et al., (1995) Science 267, 1449-1456]. It has been
reported that agonistic mouse monoclonal antibodies that bind
specifically to Fas exert apoptosis-inducing activity against
carcinoma cells to the same level as that exerted by TNF-.alpha.
[Yonehara, et al., (1989) J. Exp. Med. 169, 1747-1756].
[0008] These TNF family molecules transmit signals into cells by
binding to specific receptors on the cell surfaces. Plural
receptors for TNF family molecules are known, and they are referred
to as TNF receptor family molecules.
[0009] TNF receptor family molecules are defined by the presence of
cysteine-rich repetition of an extracellular domain. Among them,
Fas and TNFR1, which are receptors of a Fas ligand and a
TNF-.alpha., contain within the cells a region referred to as a
"death domain" sharing homology with reaper, a Drosophila suicide
gene [see Golstein, P., et al. (1995) Cell 81, 185-186; see White,
K., et al. (1994) Science 264, 677-683] and such death domain is
essential for signal transduction for apoptosis. Activation of Fas
promotes the association of an adapter molecule FADD/MORT1
containing the death domain, and induces the activation of
caspase-8 bound to FADD/MORT1. The activated caspase-8 activates
downstream caspase molecules in sequence, thereby finally leading
the cells to apoptosis [see Nagata, S., (1997) Cell 88,
355-365].
[0010] Recently, a novel TNF family molecule that induces apoptosis
has been found. Wiley et al., [see Immunity (1995) 3, 673-682]
named the molecule "TNF-related apoptosis-inducing ligand" or
briefly "TRAIL." This molecule is also referred to as "Apo-2
ligand" or "Apo-2L" [see Pitt, R. M., et al. (1996) J. Biol. Chem.
271, 12687-12690]. For convenience, this molecule is referred to as
TRAIL in this specification.
[0011] Unlike the Fas ligand, TRAIL is detected at a significant
level in many human tissues (e.g., spleen, lungs, prostate, thymus,
ovary, small intestine, large intestine, peripheral blood
lymphocyte, placenta and kidney). TRAIL is constitutively
transcribed in some cell lines. TRAIL has also been shown to
rapidly activate apoptosis at a significantly faster pace than that
induced by TNF, within a time frame resembling death signal
transduction by Fas [see Marsters, S. A., et al., (1996) Curr.
Biol. 6, 750-752].
[0012] Now 5 proteins have already been identified as TRAIL
receptors. Two receptors, TRAIL-R1 (also referred to as DR4) and
TRAIL-R2 (also referred to as DR5), have both been reported to have
death domains within the intracellular regions. The transcript of
TRAIL-R1 is recognized in many human tissues including the spleen,
peripheral blood leukocytes, small intestine and the thymus. The
transcript of TRAIL-R2 has been detected in many tissues including
the spleen, peripheral blood lymphocytes and the ovary [see Pan,
G., et al. (1997) Science 276, 111-113; see Pan, G., et al. (1997)
Science 277, 815-818 ; see Walczak, H., et al. (1997) EMBO J 16
(17) 5386-5397].
[0013] The presence of the two forms of TRAIL-R2 resulting from
alternative splicing and the high expression amount of TRAIL-R2
comprising 440 amino acids in carcinoma cells has been reported
[see Screaton, G. R., et al., (1997) Curr Biol 7 (9), 693-696 ; see
Arai, T., et al., (1998) Cancer Letters 133, 197-204].
[0014] Recombinant human TRAIL is a recombinant protein comprising
the extracellular region of TRAIL, and has been reported to induce
apoptosis in many types of carcinoma cells [see Griffith, T. S., et
al. (1998) Curr. Opin. Immunol., 10, 559-563].
[0015] Furthermore, the recombinant human TRAIL has exerted an
anti-tumor effect on a tumor-bearing mouse model using human colon
carcinoma cells and breast carcinoma cells [see Walczak, H., et al.
(1999) Nature Medicine 5, 2, 157-163]. Unlike TNF-.alpha. or FAS
ligands also belonging to the TNF receptor family and having
apoptosis-inducing activity, TRAIL did not provide damage to the
normal tissues of mice or cynomolgus monkeys [see Ashkenazi, A., et
al. (1999) J. Clin. Invest. 104, 155-162].
[0016] Based on these reports, it is thought that TRAIL selectively
induces death in tumor cells. However, such selectivity has not yet
been supported theoretically since TRAIL receptors are also
expressed in normal cells. Moreover, the recombinant human TRAIL
has recently been reported to induce apoptosis in normal human
hepatocytes [see Jo, M., et al. (2000) Nature Medicine 6, No.5,
564-567] and reported to induce apoptosis also in human brain cells
[see Nitsch, R., et al. (2000) The Lancet 356, 827-828]. Because
agonistic anti-Fas antibodies, which induce apoptosis in
hepatocytes, induce fulminant hepatitis in a very short time and
thus cause death in mice and chimpanzees, cell death induction by
TRAIL on hepatocytes has attracted attention as a particularly
significant issue. The safety of using TRAIL as a pharmaceutical
product for humans has been questioned [see Nagata, S., (2000)
Nature Medicine 6, 5, 502-503].
[0017] It has also been reported that the presence or absence of
the cell-death-inducing activity of TRAIL on hepatocytes depends on
the type of recombinant TRAIL protein [see Lawrence, D., et al.
(2001) Nature Medicine 7, 4, 383-385]. However, the safety of the
recombinant TRAIL protein is still being studied.
[0018] Recently, anti-Fas antibodies that do not induce hepatopathy
when administered to mice have been reported for the first time
[see Ichikawa, K., et al. (2000) International Immunology 12, No.4,
555-562]. There have been no known recombinant Fas ligands
confirmed not to induce hepatopathy. This suggests that antibodies
having activity that may be unavailable from ligands can be
obtained. However, the theoretical background of the reason that
the antibodies show no hepatotoxicity in spite of inducing
apoptosis in T cells has not been revealed. For example, in the
case of a different antigen such as TRAIL, it has not been
demonstrated whether or not agonistic antibodies having no toxicity
can be obtained.
[0019] TRAIL binds to TRAIL-R1, TRAIL-R2, or both, and induces
apoptosis. However, via which receptor the signals to induce
apoptosis in hepatocytes are introduced by TRAIL has not been
shown. Furthermore, no research has been done based on the idea of
whether hepatotoxicity can be avoided by adding TRAIL-R1/R2
selectivity to agonistic antibodies.
[0020] An effective therapeutic means against malignant tumors
involves removing carcinoma cells and protecting normal tissues or
cells. A drug whose action mechanism is apoptosis induction by the
recombinant human TRAIL may cause damages to normal tissues,
particularly the liver and the brain, even if it is able to remove
carcinoma cells.
[0021] Currently, monoclonal antibodies such as a chimeric antibody
targeting CD20, which is a receptor present on the cell membrane,
and a humanized antibody targeting Her2/neu are used against
malignant tumors as target diseases, and their therapeutic effects
have been recognized [see Kensei Tobinai (2003) Molecular Medicene
40, 10, 1176-1181; see Masahiro Takada et al. (2003) Molecular
Medicine 40, 10 1166-1174]. Since antibodies have characteristics
including a long half-life in blood and high specificity to
antigens, they are particularly useful as anti-tumor agents. For
example, in the case of antibodies targeting tumor-specific
antigens, the administered antibodies are assumed to accumulate in
tumors. Thus, attack against carcinoma cells by the immune system
can be expected by complement-dependent cytotoxicity and
antibody-dependent cell-mediated cytotoxicity. In addition, the
binding of a drug such as a radionuclide, a cytotoxic substance or
the like to the antibodies enables the efficient delivery of the
drug bound to the antibody to tumor sites. At the same time,
reduced side effects can be expected due to decreased amounts of
the drug having reached other non-specific tissues. When
tumor-specific antigens have activity to induce cell death,
antibodies having agonistic activity are administered, and when
tumor-specific antigens are involved in cell proliferation and
survival, antibodies having neutralization activity are
administered. And then, the accumulation of tumor-specific
antibodies and suppression of tumor growth or regression of tumors
due to the activity of the antibodies can be expected.
[0022] It is thought to be appropriate to apply antibodies as
anti-tumor agents because of the characteristics described above.
In addition, if antibodies are those against TRAIL receptors,
antibodies that may be obtained can avoid causing damage to the
liver, which is unable to avoid with the recombinant human TRAIL,
and have equivalent apoptosis-inducing activity against carcinoma
cells.
[0023] In addition to these reports, recently, it has been reported
that monoclonal antibodies against TRAIL-R1 and TRAIL-R2 induce
apoptosis in carcinoma cells like a recombinant human TRAIL [see
Griffith, T. S., et al. (1999) J. Immunol. 162, 2597-2605; see
Chuntharapai, A., et al. (2001) J. Immunol. 166, 4891-4898; see
Mori, E., et al (2003) Cell Death Differ. in press]. These
antibodies take anti-tumor effect on tumor-bearing mice constructed
by using human colon carcinoma cells [see Chuntharapai, A., et al.
(2001) J. Immunol. 166, 4891-4898].
[0024] With respect to anti-tumor mechanism by an antibody, various
mechanisms according to a characteristic of a target molecule have
been made clear. For example, exclusion by an immune system of a
host cell such as complement dependent cytotoxicity (CDC) and
antibody dependent cell mediated cytotoxity(ADCC), and the
mechanism in which the suppression of cell growth and the cell
death are induced directly through a target molecule on the surface
of a tumor cell have been reported. In the mechanisms in which a
tumor cell is directly acted on, there are an example in which only
binding of antibody to a target molecule can induce a signal
[Schneider, H., et al. (1997) Blood 89, 473-482] and an example in
which only binding of antibody to a target molecule cannot induce a
signal [Xu, Y., et al. (2003) J. Immunol., 171, 562-568]. In the
latter example, it is known that a signal is not induced until
antibodies which are bound to a target molecule are cross-linked (a
substance which cross-links antibodies is referred to as a
cross-linker).
[0025] It has been suggested as a result of experiments using mice
as a model system that in case of anti-CD20 antibody or
anti-Her2/neu antibody, ADCC through a Fc receptor which is
expressed on the surface of immunocompetent cells exerts the
anti-tumor activity [see Clynes, R. A., et al. (2000) Nat Med., 6,
4, 443-446]. Furthermore, it has been found that the anti-tumor
activity of anti-CD20 antibody is enhanced when the antibody is
cross-linked [see Ghetie, M-A., et al. (2001) Blood, 97, 1392-1398]
and it has been suggested that the antibody exhibits directly the
activity of the growth suppression against tumor cells.
[0026] Anti-tumor effect of anti-CD20 antibody and so on is taken
by a direct growth supressin of tumor cells caused by ADCC, CDC and
cross-linking through immunocompetent cells as described above. It
is thought that the anti-tumor effect is not taken by an antibody
only but through an exogenous factors in any case. Therefore, it is
assumed that the therapeutic effect depends on a condition of a
host. In contrast, in case of anti-TRAIL-R antibody, it was assumed
that binding of an antibody to a receptor induced the transduction
of an apoptosis signal in a cell. Accordingly, it was thought
firstly that anti-tumor effect taken by an antibody only without
the mediation of exogenous factors was expected. However, as a
result of in vitro experiments using anti-TRAIL-R antibody, it was
reported that the exertion of the anti-tumor activity by the
induction of cell death needs cross-linking of antibodies [see
Griffith, T. S. et al. (1999) J. Immunol. 162, 2597-2605; see
Chuntharapai, A., et al. (2001) J. Immunol. 166, 4891-4898; see
Mori, E., et al. (2003) Cell Death Differ. in press]. This suggests
that the activity is affected by exogenous factors.
[0027] On the contrary, anti-TRAIL-R antibody which can induce cell
death by antibody only has been reported [see WO98/51793]. We have
studied the activity of the antibody by using a purified antibody,
and it was confirmed that the treatment with the antibody only did
not exert the cell-death-inducing activity. It is known that
antibodies are generally polymerized in a condition that antibodies
are secreted into a culture supernatant of antibody producing cells
and during a purification process. Accordingly, monomer antibodies
and polymerized antibodies co-exist in antibody solution. It is
thought that these polymerized antibodies, which have a plenty of
functional antigen binding sites like cross-linked antibodies but
unlike monomer antibodies, form a polymer of antigens which bound
to antibodies. Since TRAIL-R transduces a cell-death-inducing
signal when it is polymerized, a polymer of anti-TRAIL-R antibodies
can form a polymer of TRAIL-R by themselves, a polymer of
anti-TRAIL-R antibodies can induce cell death on carcinoma cells.
If the treatment by antibodies only induces cell death, it cannot
been determined whether a monomer exerts the inducing activity or a
polymer exerts the inducing activity as long as a polymer is
included in the antibodies.
[0028] The antibody polymer can be removed by increasing the purity
of monomer antibody by an operation of a purification method such
as gel filtration. Since a polymer antibody induces the formation
of aggregates and may possibly cause adverse side effects, a
polymer antibody is generally removed as much as possible. Thus, in
case where a polymer alone possesses the activity, the activity is
lost by the increase of the purity of a monomer antibody. Thus the
activity of a polymer antibody cannot be said as the activity
exerted by the antibody only. Consequently, it is said that the
antibody in which only a polymer has the activity is the antibody
which need factors other than the antibody such as a cross-linker
to exert the activity.
[0029] In contrast, since an antibody which needs cross-linking to
exert the activity in vitro can take anti-tumor effect on tumor
bearing mice when it is administered alone into the mice, it is
assumed that there existed a factor which can cross-link antibodies
administered in a mouse body. The factor includes a Fc receptor and
a complement.
[0030] It has been suggested that a complement can act as a
cross-linker by in vitro experiments [see Chuntharapai, A., et al.
(2001) J. Immunol. 166, 4891-4898]. It is assumed that the
complement has the same functions in a human body. However,
regarding the complement, there are disorders caused by a
complement deficiency or abnormality of a complement [see M.
Kondoh, S. Takemura. (1994) Hotai to Sono Rinsho "Complement and
its clinical study" 99-102]. For a patient suffering from these
disorders, it is not expected that a complement can act as a
cross-linker.
[0031] An Fc receptor is expressed on an effecter cell such as T
cell, NK cell, neutrophil, macrophage. It is known that the
receptor activates an effecter cell when it recognizes an antibody
which is bound to a target cell and is involved in ADCC which
causes lysis of a target cell. It has been reported that there is a
polymorphism in Fc receptors and the binding affinity to Fc region
of an antibody differs among a type in the polymorphism [see
Radaev, S. et al. (2001) Mol. Immunol. 38, 1073-1083]. It is also
known that the number of effecter cells expressing Fc receptors
decreases as a side effect of anti-tumor agent. Thus, it has been
reported that, in some of patients to whom an anti-tumor agent is
administered, the number of cells expressing Fc receptors such as
neutrophils decreases compared to a patient who is not administered
with anti-tumor agent [See TOKYO METROPOLITAN KOMAGOME HOSPITAL,
Chemotherapy, 2001, A method of using anti-tumor drug errorlessly,
141-153]. Considering these reports, the action of Fc receptors is
not always expected. Under these circumstances, an antibody which
needs exogenous factors to exert its activity cannot always take
anti-tumor effects.
[0032] Accordingly, it is said that an antibody of which monomer
alone can cross-link molecules on the surface of cells to transduce
a signal is useful for the treatment of tumor since its activity is
not affected by other factors. Thus attempts to produce various
modified antibodies which have the activity not affected by other
factors such as an antibody having more than three
antigen-recognizing sites have been undertaken [see JP Patent
Publication (PCT Translation) No. 2003-531588]. However, since
these antibodies have problems that include a shortening of a half
life in blood, antigenicity and the like, and the usefulness as a
pharmaceutical antibody may be lost, there are many problems to be
solved.
[0033] We have tried to obtain anti-TRAIL-R2 antibody which can
transduce an apoptosis signal as a monomer, and finally obtained
the antibody of the present invention. It is assumed that the
present antibody is appropriate to be used as an anti-tumor agent
because of its characteristics as described above. Furthermore,
since the present antibody has the activity when used as a monomer,
the antibody not only maintains the usefulness as a therapeutic
antibody but also exerts its activity independently of exogenous
factors.
SUMMARY OF THE INVENTION
[0034] A first purpose of the present invention is to provide a
novel antibody or a molecule analogous thereto, which is capable of
binding to human TRAIL-R1 and/or human TRAIL-R2 and induces
apoptosis specifically in carcinoma cells, without inducing damage
to normal human hepatocytes to which a recombinant human TRAIL
protein can cause damages. The novel antibody or a molecule
analogous thereto also includes an antibody or a molecule analogous
thereto which is capable of inducing apoptosis independently of
exogenous factors and as a monomer in carcinoma cells. A second
purpose of the present invention is to provide a prophylactic or
therapeutic agent comprising the above antibody or a molecule
analogous thereto as an active ingredient against various malignant
tumors including solid tumors that are currently difficult to
treat.
[0035] As a result of intensive studies on the production of
antibodies against human TRAIL-R1 and -R2, we have succeeded in
obtaining monoclonal antibodies from the culture supernatant by
immunizing transgenic mice capable of producing human antibodies by
genetic engineering techniques with human TRAIL-R1 or R2,
generating hybridomas producing novel monoclonal antibodies that
bind to TRAIL-R1 and/or TRAIL-R2 using the method of Kohler and
Milstein et al. [see (1975) Nature 256, 495], which is generally
used in monoclonal antibody production.
[0036] Furthermore, we have completed the present invention by
finding that the novel monoclonal antibodies induce apoptosis
specifically in carcinoma cells by binding to TRAIL-R1 and/or R2
present on the surfaces of carcinoma cells. Furthermore, we have
completed the present invention by finding that the novel
monoclonal antibodies induce apoptosis independently of exogenous
factors and as a monomer specifically in carcinoma cells by binding
to TRAIL-R1 and/or R2 present on the surfaces of carcinoma
cells.
[0037] The present invention is as follows.
[0038] (1) An antibody or a functional fragment thereof, binding to
TRAIL-R1 and/or TRAIL-R2.
[0039] The above antibody or the functional fragment thereof has at
least one property selected from the following (a) to (c) of:
[0040] (a) having activity to induce apoptosis in carcinoma cells
expressing TRAIL-R1 and/or TRAIL-R2;
[0041] (b) not having effect on normal human cells expressing
TRAIL-R1 and/or TRAIL-R2; and
[0042] (c) not inducing human hepatocyte toxicity.
[0043] In the present invention, an antibody or a functional
fragment thereof having all the above properties (a) to (c) is
preferred. Furthermore, the antibody or the functional fragment
thereof of the present invention also includes an antibody or a
functional fragment thereof that has at least one property of the
above (a) to (c), and (1) binds to TRAIL-R2, but does not bind to
TRAIL-R1, or (2) binds to both TRAIL-R2 and TRAIL-R1.
[0044] The above antibody or the functional fragment thereof also
includes an antibody or a functional fragment thereof which has
activity to induce apoptosis in carcinoma cells expressing TRAIL-R1
and/or TRAIL-R2 independently of exogenous factors and as a
monomer.
[0045] (2) The above antibody is a monoclonal antibody produced by
a mouse-mouse hybridoma, such as E-11-13, H-48-2, L-30-10, N-18-12,
W-40-5, X-14-4, X-51-12, F-4-8, G-3-10, 0304, 0322 or KMTR1, and is
preferably a human antibody. The type of the monoclonal antibody
produced by E-11-13, H-48-2, L-30-10, N-18-12, W-40-5, X-14-4,
X-51-12, F-4-8, 0304, 0322 or KMTR1 is the immunoglobulin G(IgG),
and the type of the monoclonal antibody produced by G-3-10 is the
immunoglobulin M(IgM). H-48-2, E-11-13, F-4-8, L-30-10, 0304 and
KMTR1 of the above hybridomas are respectively deposited
internationally, and the desposition information is as follows.
1 Name Accession No. Deposition date Deposited with: H-48-2 FERM
BP-7599 May 18, 2001 International Patent E-11-13 FERM BP-7698 Aug
8, 2001 Organism Depositary, FERM BP-7770 Oct 11, 2001 National
Institute of F-4-8 FERM BP-7699 Aug 8, 2001 Advanced Industrial
FERM BP-7768 Oct 11, 2001 Science and Technology L-30-10 FERM
BP-7700 Aug 8, 2001 (Central 6, 1-1-1, FERM BP-7769 Oct 11, 2001
Higashi, Tsukuba, 0304 FERM BP-8037 May 10, 2002 Ibaraki, Japan)
KMTR1 FERM BP-8038 May 10, 2002
[0046] Among these hybridomas, 0304 and 0322 produce an antibody
which has the activity to induce apoptosis independently of
exogenous factors and as a monomer.
[0047] Examples of carcinoma cells include colon carcinoma cells,
Colo205, glioma U251 cells and T cell lymphoma Jurkat cells. The
carcinoma cells are appropriately selected from these cells.
[0048] (3) The antibody or the functional fragment thereof of the
present invention has, under conditions where the number of cells
is 7.5.times.10.sup.4 and the reaction time is 24 hours, an LD50
value for human hepatocytes of 0.01 .mu.g/ml or more, preferably
0.1 .mu.g/ml or more, further preferably 2 to 10 .mu.g/ml, still
further preferably 10 to 100 .mu.g/ml, or most preferably 10
.mu.g/ml or more (e.g., 100 .mu.g/ml or more). In the meantime, the
antibody or the functional fragment thereof of the present
invention has, under conditions where the number of cells is
2.5.times.10.sup.3 and the reaction time is 48 hours, an LD50 value
for carcinoma cells (e.g., Colo205 cells, U251 cells or Jurkat
cells) of 100 .mu.g/ml or less, preferably 10 .mu.g/ml or less,
more preferably 0.7 .mu.g/ml or less, further preferably 0.02 to
0.11 .mu.g/ml, or most preferably 0.02 .mu.g/ml or less. Moreover,
the antibody or the functional fragment thereof that is
particularly preferred in the present invention has a combination
of LD50 values, one of which is between 2 and 100 .mu.g/ml for
human heptocytes under conditions where the number of cells is
7.5.times.10.sup.4 and the reaction time is 24 hours, and the other
of which is between 0.02 and 0.11 .mu.g/ml for carcinoma cells
under conditions where the number of cells is 2.5.times.10.sup.3
and the reaction time is 48 hours.
[0049] The above LD50 values of the antibody of the present
invention for hepatocytes or carcinoma cells are obtained by
measurement with a reaction volume of 110 to 120 .mu.l per reaction
system (per well).
[0050] (4) Furthermore, the antibody or the functional fragment
thereof of the present invention has an LD50 value for human
hepatocytes under conditions where the number of cells is
7.5.times.10.sup.4 and the reaction time is 24 hours that is 2
times or more, preferably 10 times or more, more preferably 50
times or more (e.g., 50 times to 100 times), further preferably 100
times or more (e.g., 100 times to 250 times), still further
preferably 250 times to 1000 times, or most preferably 1000 times
or more greater than that for carcinoma cells under conditions
where the number of cells is 2.5.times.10.sup.3 and the reaction
time is 48 hours.
[0051] (5) Furthermore, the antibody or the functional fragment
thereof of the present invention can suppress the growth of tumors
(e.g., those derived from Colo205 cells transplanted to nude mice)
or regress tumors. In this case, a period during which tumor cell
proliferation can be suppressed, or during which tumor regression
can be achieved when the antibody or the functional fragment
thereof of the present invention is administered, is at least 9
days, preferably at least 11 days or further preferably at least 13
days. Hereinafter, the period, in order of preference, is as
follows: at least 30 days, at least 60 days, and most preferably at
least 120 days. In addition, the dose of the antibody or the
functional fragment thereof of the present invention that is
administered to a tumor-bearing animal to be tested (e.g., a body
weight of a tumor-bearing experimental animal is 20 g) is between
0.1 .mu.g/body (5 .mu.g/kg) and 100 .mu.g/body (5 mg/kg). For
example, the dose is 100 .mu.g/body or 5 mg/kg, preferably 20
.mu.g/body or 1 mg/kg, more preferably 4 .mu.g/body or 200
.mu.g/kg, or further preferably 1 .mu.g/body or 50 .mu.g/kg. A dose
of 0.5 .mu.g/body (25 .mu.g/kg) may also be administered. The
administration frequency is, for example, once to 3 times per week,
or administration is performed on alternate days.
[0052] Moreover, the anti-tumor effect of the antibody (e.g., 0304
antibody or E-11-13 antibody) or the functional fragment thereof of
the present invention in tumor-bearing mice is as follows.
[0053] (a) When administered at a concentration of 20 .mu.g/mouse
to a 4- to 6-week-old tumor-bearing mouse having a 100 mm.sup.3
tumor, the antibody or the functional fragment thereof can induce
an average of 14% or more tumor reduction by 4 days after the
initial administration. In this case, an average of 14% or more
tumor reduction can be maintained for at least 7 days.
[0054] (b) When administered at a concentration of 20 .mu.g/mouse
to a 4- to 6-week-old tumor-bearing mouse having a 100 mm.sup.3
tumor, the antibody or the functional fragment thereof can induce
an average of 65% or more tumor reduction by 4 days after the
initial administration.
[0055] (c) When administered at a concentration of 20 .mu.g/mouse
to a 4- to 6-week-old tumor-bearing mouse having a 100 mm.sup.3
tumor, the antibody or the functional fragment thereof can induce
an average of 80% or more tumor reduction by 7 days after the
initial administration. In this case, an average of 80% or more
tumor reduction can be maintained for at least 4 days.
[0056] (d) When administered at a concentration of 25 .mu.g/mouse
to a 12-week-old tumor-bearing mouse having a 100 mm.sup.3 tumors,
the antibody or the functional fragment thereof can induce an
average of 45% or more tumor reduction by 3 days after the initial
administration.
[0057] (e) When administered at a concentration of 25 .mu.g/mouse
to a 12-week-old tumor-bearing mouse having a 100 mm.sup.3 tumor,
the antibody or the functional fragment thereof can induce an
average of 65% or more tumor reduction by 5 days after the initial
administration. In this case, an average of 65% or more tumor
reduction can be maintained for at least 27 days.
[0058] (f) When administered at a concentration of 20 .mu.g/mouse
to a 4- to 6-week-old tumor-bearing mouse having a 300 mm.sup.3
tumor, the antibody or the functional fragment thereof can induce
an average of 39% or more tumor reduction by 4 days after the
initial administration. In this case, an average of 39% or more
tumor reduction can be maintained for at least 14 days.
[0059] Examples of the relevant tumor include at least one tumor
selected from the group consisting of colon cancer, colorectal
cancer, lung cancer, breast cancer, brain tumor, malignant
melanoma, renal cell carcinoma, bladder cancer, leukemia,
lymphomas, T cell lymphomas, multiple myeloma, gastric cancer,
pancreas cancer, cervical cancer, endometrial carcinoma, ovarian
cancer, esophageal cancer, liver cancer, head and neck squamous
cell carcinoma, cutaneous cancer, urinary tract carcinoma, prostate
cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer,
thecomatosis, androblastoma, endometrium hyperplasy, endometriosis,
embryoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous
hemangioma, angioblastoma, retinoblastoma, astrocytoma,
neurofibroma, oligodendroglioma, medulloblastoma,
ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma,
osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma and Wilms
tumor.
[0060] (6) An antibody or a functional fragment thereof having
amino acid sequences of the mature portions of a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma E-11-13, which are respectively represented
by SEQ ID NOS: 17 and 19; a heavy chain variable region and a light
chain variable region of the antibody produced by a hybridoma
L-30-10, which are respectively represented by SEQ ID NOS: 21 and
23; a heavy chain variable region and a light chain variable region
of the antibody produced by a hybridoma H-48-2, which are
respectively represented by SEQ ID NOS: 25 and 27; a heavy chain
variable region and a light chain variable region of the antibody
produced by a hybridoma 0304, which are respectively represented by
SEQ ID NOS: 29 and 31; or a heavy chain variable region and a light
chain variable region of the antibody produced by a hybridoma
KMTR1, which are respectively represented by SEQ ID NOS: 33 and
35.
[0061] The above antibody or the functional fragment thereof has
amino acid sequences of the mature portions of a heavy chain
variable region and a light chain variable region that are encoded
by nucleic acid sequences isolated from a hybridoma E-11-13, which
are respectively represented by SEQ ID NOS: 16 and 18; a heavy
chain variable region and a light chain variable region that are
encoded by nucleic acid sequences isolated from a hybridoma
L-30-10, which are respectively represented by SEQ ID NOS: 20 and
22; a heavy chain variable region and a light chain variable region
that are encoded by nucleic acid sequences isolated from a
hybridoma H-48-2, which are respectively represented by SEQ ID NOS:
24 and 26; a heavy chain variable region and a light chain variable
region that are encoded by nucleic acid sequences isolated from a
hybridoma 0304, which are respectively represented by SEQ ID NOS:
28 and 30; or a heavy chain variable region and a light chain
variable region that are encoded by nucleic acid sequences isolated
from a hybridoma KMTR1, which are respectively represented by SEQ
ID NOS: 32 and 34.
[0062] (7) A hybridoma producing monoclonal antibodies that bind to
TRAIL-R2, which is selected from the group consisting of E-11-13,
H-48-2, L-30-10, N-18-12, W-40-5, X-14-4, X-51-12, F-4-8, G-3-10,
0304, 0322 and KMTR1.
[0063] (8) A method for producing anti-TRAIL-R2 monoclonal
antibodies, comprising culturing the above hybridoma and collecting
the antibodies binding to TRAIL-R2 from the obtained culture
product.
[0064] (9) A method for producing anti-TRAIL-R2 monoclonal
antibodies, comprising isolating a gene encoding a monoclonal
antibody from the above hybridoma, constructing an expression
vector having the gene, introducing the expression vector into a
host to express the above monoclonal antibody, and collecting
anti-TRAIL-R2 monoclonal antibodies from the host, or the culture
supernatant or the secretion of the obtained host.
[0065] Examples of a host include any host selected from the group
consisting of Escherichia coli, yeast cells, insect cells,
mammalian cells and plant cells, and mammals.
[0066] (10) A prophylactic or therapeutic agent against tumors,
comprising as an active ingredient the above antibody or the
functional fragment thereof.
[0067] Examples of the tumor include at least one tumor selected
from the group consisting of colon cancer, colorectal cancer, lung
cancer, breast cancer, brain tumor, malignant melanoma, renal cell
carcinoma, bladder cancer, leukemia, lymphomas, T cell lymphomas,
multiple myeloma, gastric cancer, pancreas cancer, cervical cancer,
endometrial carcinoma, ovarian cancer, esophageal cancer, liver
cancer, head and neck squamous cell carcinoma, cutaneous cancer,
urinary tract carcinoma, prostate cancer, choriocarcinoma,
pharyngeal cancer, laryngeal cancer, thecomatosis, androblastoma,
endometrium hyperplasy, endometriosis, embryoma, fibrosarcoma,
Kaposi's sarcoma, hemangioma, cavernous hemangioma, angioblastoma,
retinoblastoma, astrocytoma, neurofibroma, oligodendroglioma,
medulloblastoma, ganglioneuroblastoma, glioma, rhabdomyosarcoma,
hamartoblastoma, osteogenic sarcoma, leiomyosarcoma, thyroid
sarcoma and Wilms tumor.
[0068] The present invention is explained in detail as follows.
This specification includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application No. 2001-150213 (filed on May 18, 2001), Japanese
Patent Application No. 2001-243040 (filed on Aug. 9, 2001), and
Japanese Patent Application No. 2001-314489 (filed on Oct. 11,
2001) which are priority documents of the present application.
[0069] The anti-TRAIL-R1 and R2 monoclonal antibodies have been
reported to have activity to induce apoptosis in carcinoma cells
[see Griffith, T. S., et al. (1999) J. Immunol. 162, 2597-2605; see
Chuntharapai, A., et al. (2001) J. Immunol. 166, 4891-4898].
However, these antibodies are derived from mice. In addition, the
cytotoxicity against normal human hepatocytes, which is also a
problem in a recombinant human TRAIL protein, is a concern.
Moreover, it has been revealed that a cross-linker is needed for
the activity to induce apoptosis. A cross-linker is a substance
which can cross-link antibodies. The antibodies are cross-linked by
the cross-linker and become a complex of plural antibodies and the
complex has plural antigen binding sites. By the action of the
complex having plural binding sites, TRAIL-Rs on the surface of the
cells aggregate and an apoptosis signal is transduced in the cells
and the apoptosis of the cells are induced. A cross-linker includes
a Fc receptor and a complement.
[0070] Surprisingly, the novel human anti-TRAIL-R2 monoclonal
antibody of the present invention has been revealed to have no side
effect of inducing cytotoxicity against not only cells derived from
a normal human tissue, but also normal hepatocytes for which
cytotoxicity by the recombinant human TRAIL protein is a concern.
We have obtained a novel anti-TRAIL-R2 monoclonal antibody. That
is, we have completed the present invention by succeeding for the
first time in the world in producing a novel monoclonal antibody
provided with possible advantages of improved safety and
therapeutic effects. The monoclonal antibody is preferably a human
antibody. Its antigenicity, which is always a problem in the case
of a mouse-derived antibody, has already been avoided.
[0071] Any antibody type of immunoglobulin G(IgG), A(IgA), E(IgE)
or M(IgM) can be appropriately used as the antibody. Normally, IgG
is more preferred.
[0072] The present invention is explained in detail by making clear
the meanings of the words and phrases used in the present invention
as follows.
[0073] 1. TRAIL and the Antibody
[0074] The antibody of the present invention is an antibody against
the receptor of a tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) (TRAIL-R). The antibodies of the
present invention are (1) an antibody reacting with TRAIL-R1, (2)
an antibody reacting with TRAIL-R2, and (3) an antibody reacting
with both TRAIL-R1 and TRAIL-R2. In the present invention, the
antibody (1) may be referred to as "the anti-TRAIL-R1 antibody,"
and the antibodies (2) and (3) may be referred to as "the
anti-TRAIL-R2 antibodies." In addition, when both TRAIL receptors,
TRAIL-R1 and TRAIL-R2, are conveniently explained together in this
specification, they may be referred to as "TRAIL-R1 and R2."
Therefore, for example, the description of "TRAIL-R1 and R2
expression vectors" (see Example 1, below) is meant to explain two
expression vectors, the expression vector of TRAIL-R1 and the
expression vector of TRAIL-R2.
[0075] The "antibody" in the present invention is an antibody or a
part thereof having reactivity to the human TRAIL-R1 and R2 or a
part thereof as defined above, and includes functional fragments of
these antibodies. The "functional fragment" means a part (partial
fragment) of the antibody retaining one or more actions of the
antibody on an antigen. Specific examples of functional fragments
include F(ab').sub.2, Fab', Fab, Fv, disulfide-bound Fv, single
chain Fv(scFv) and the polymers thereof (D. J. King., Applications
and Engineering of Monoclonal Antibodies.,1998 T. J. International
Ltd).
[0076] The "monomer of an antibody" or "monomer antibody" of the
present invention means an antibody which is present in a fraction
in which a monomer is clearly eluted when antibody proteins are
subjected to a general biochemical method for separating and
purifying a protein such as gel filtration. For example, the
fraction is a fraction in which a monomer antibody is eluted
clearly by comparing a molecular weight with molecular weight
markers and considering a deduced molecular weight of an
antibody.
[0077] A fragment of the "monomer of an antibody" or "monomer
antibody"which is bound to TRAIL-R is included in the "monomer of
an antibody" or "monomer antibody". The "monomer of an antibody" or
"monomer antibody" can be said to be an "antibody as a single
substance" or "single antibody", and "to use antibody as a monomer"
can be said as "to use antibody as a single substance" or "to use
antibody in the form of a single substance". An antibody which is
not the "monomer of an antibody" or "monomer antibody" includes an
antibody in which plural antibodies form a complex, and is said as
a "polymer (multimer) of antibodies" or "polymer (multimer)
antibody". The complex formed by plural antibodies can be formed by
the cross-linker mentioned above or by the polymerization of two or
more of antibody molecules. When the "monomer of an antibody" or
"monomer antibody" is used, it can consist of a mixture with the
"polymer (multimer) of antibodies" or "antibody polymer
(multimer)". In this case, the mixture contains the "polymer
(multimer) of antibodies" or "antibody polymer (multimer)" by 0.5%
or less, preferably 0.2% or less, more preferably 0.1% or less.
Most preferably, the "polymer (multimer) of antibodies" or
"antibody polymer (multimer)" is not contained.
[0078] The "exogenous factor" or "factor present exogenously" of
the present invention is a factor other than an antibody which is
necessary for the exertion of the activity of the antibody. The
"exogenous factor" or "factor present exogenously" includes a
complement, Fc receptor, cell expressing Fc receptor and so on. It
also includes the cross-linker above. The "monomer of an antibody"
or "monomer antibody" of the present invention can bind to TRAIL-R
and transduce a signal in a cell expressing TRAIL-R to induce an
apoptosis in the cell even in the absence of the "exogenous factor"
or "factor present exogenously".
[0079] That is to say, the "monomer of an antibody" or "monomer
antibody" of the present inventionis an antibody which has the
activity to induce apoptosis in a cell without depending on
(independently of) the "exogenous factor" or "factor present
exogenously".
[0080] The "human antibody" in the present invention means an
antibody which is the expression product of a human-derived
antibody gene.
[0081] Examples of the antibody of the present invention include
various antibodies having a property of inducing apoptosis in
carcinoma cells expressing the human TRAIL-R1 and R2 as later
described in Example 7.
[0082] Examples of the antibody of the present invention also
include various antibodies having a property of inducing apoptosis
as a monomer in carcinoma cells expressing the human TRAIL-R1 and
R2 as later described in Example 28.
[0083] The antibody of the present invention encompasses a
monoclonal antibody comprising heavy chains and/or light chains
having amino acid sequences with deletion, substitution or addition
of one or a plurality of amino acids in each amino acid sequence of
the heavy chain and/or light chain of the antibody. The
above-described partial amino acid alteration (deletion,
substitution, insertion or addition) can be introduced into the
amino acid sequence of the antibody of the present invention by,
for example, a method which involves partial alteration of the
nucleotide sequence encoding the amino acid sequence. The partial
alteration can be introduced into the nucleotide sequence by a
standard method using known site-specific mutagenesis (Proc Natl
Acad Sci USA., 1984 Vol 81: 5662). Here, the antibody is an
immunoglobulin wherein all the regions, including a heavy chain
variable region and a heavy chain constant region, and a light
chain variable region and a light chain constant region composing
the immunoglobulin, are derived from a gene encoding the
immunoglobulin.
[0084] The antibody of the present invention also encompasses
antibodies having any immunoglobulin classes and isotypes.
[0085] The anti-TRAIL-R1 and R2 antibodies of the present invention
can be produced by the following production method. Specifically,
for example, the above-defined human TRAIL-R1 and R2 or a part
thereof is bound to an appropriate substance (e.g., bovine serum
albumin) for enhancing the antigenicity of an antigen, and then
non-human mammals including human antibody-producing transgenic
mice and the like are immunized with the bound product, together
with an immunopotentiator (e.g., Freund's complete or incomplete
adjuvant) if necessary. Alternatively, immunization can also be
performed by introducing a gene encoding the human TRAIL-R1 or
human TRAIL-R2, and then administering animal cells excessively
expressing TRAIL-R1 or TRAIL-R2 on the cell surfaces. Monoclonal
antibodies can be obtained by culturing hybridomas that are
obtained by fusing antibody-producing cells obtained from immunized
animals with myeloma cells incapable of producing autoantibodies,
and then selecting clones that produce monoclonal antibodies
showing specific affinity for the antigens used for
immunization.
[0086] The antibody of the present invention encompasses an
antibody converted to have a different subclass by alteration using
genetic engineering techniques known to a person skilled in the
art. For. example, the subclass switching of the antibody of the
present invention to IgG2 or IgG4 enables antibodies with a low
binding activity to Fc receptors to be obtained. Also, the subclass
switching of the antibody of the present invention to IgG1 or IgG3
enables antibodies with a high binding activity to Fc receptors to
be obtained. Moreover, the binding activity to a Fc receptor can
also be changed by artificially altering the amino acid sequence of
the constant region of the antibody of the present invention, or by
binding with a constant region sequence having such an altered
sequence. Furthermore, the therapeutic effect against diseases such
as cancer can be further enhanced by binding to the antibody of the
present invention a radionuclide such as iodine, yttrium, indium or
technitium, (J. W. Goding, Monoclonal Antibodies: principles and
practice., 1993 Academic Press), bacterial toxin such as pyocyanic
toxin, diphteria toxin or lysin, chemotherapeutics such as
methotrexate, mitomycin or calicheamicin (D. J. King, Applications
and Engineering of Monoclonal Antibodies., 1998 T. J. International
Ltd.; M. L. Grossbard., Monoclonal Antibody-Based Therapy of
Cancer., 1998 Marcel Dekker Inc), or else a prodrug such as
Maytansinoid (Chari et al., Cancer Res., 1992 Vol. 52: 127; Liu et
al., Proc. Natl. Acad. Sci. USA, 1996 Vol. 93: 8681).
[0087] Moreover, we have found that the antibodies of the present
invention having the property of binding to TRAIL-R2 but not the
property of binding to TRAIL-R1 include antibodies that do not
induce human hepatocyte toxicity. Therefore, the present invention
also provides a method for producing anti-TRAIL-R2 antibodies
having no hepatocyte toxicity, comprising a step of selecting
antibodies that do not bind to TRAIL-R1 from the antibody
population that binds to TRAIL-R2. However, the antibody of the
present invention having no hepatocyte toxicity is not limited to
an antibody having the property of binding to TRAIL-R2 but not the
property of binding to TRAIL-R1.
[0088] The present invention encompasses the following operation
steps in monoclonal antibody production. Specifically, the steps
are, for example: (1) purification of biopolymers and/or the
preparation of cells excessively expressing antigen proteins on the
cell surfaces (these biopolymers and/or cells are used as
immunogens); (2) immunization of animals by the injection of an
antigen, blood collection, testing of the antibody titer, and
determination of a time for excising the spleen and the like
followed by preparation of antibody-producing cells; (3)
preparation of myeloma cells (hereinafter referred to as
"myeloma"); (4) cell fusion of the antibody-producing cells with
myeloma, (5) selection of a hybridoma group producing a target
antibody; (6) division into a single cell clone (cloning); (7) if
necessary, culture of hybridomas for producing monoclonal
antibodies in large quantities, or breeding of animals having the
hybridomas transplanted therein; and (8) study of the physiological
activities and the recognition specificity of the thus-produced
monoclonal antibodies, or testing of the characteristics as a
labeled reagent.
[0089] The production method of anti-TRAIL-R1 and R2 monoclonal
antibodies is described in detail according to the above steps, but
the production method of the antibody is not limited to this
method. For example, antibody-producing cells and myeloma other
than splenocytes can also be used.
[0090] (1) Purification of Antigen
[0091] As the antigen, a fusion protein of the extracellular
regions of human TRAIL-R1 and R2 with the Fc region of a human IgG
(hereinafter referred to as TRAIL-R1-hFc and TRAIL-R2-hFc) can be
used. TRAIL-R1-hFc and TRAIL-R2-hFc can be obtained by integrating
a DNA encoding a fusion protein of TRAIL-R1 or R2 with the Fc
region of a human IgG into an expression vector for animal cells,
introducing the expression vector into animal cells, and then
purifying from the culture supernatant of the obtained transfectant
strain. Alternatively, TRAIL-R1-hFc and TRAIL-R2-hFc commercially
available from ALEXIS and the like can also be used. Furthermore,
purified TRAIL-R1 and R2 from the cell membranes of a human cell
line, can also be used as the antigen. Furthermore, the primary
structures of TRAIL-R1 and R2 are known [see Pan, G., et al. (1997)
Science 276, 111-113 and Science 277, 815-818 ; see Walczak, H., et
al. (1997) EMBO J 16 (17) 5386-5397]. Thus, according to a method
known by a person skilled in the art, peptides are chemically
synthesized from the amino acid sequences of TRAIL-R1 and R2, and
then can also be used as the antigen.
[0092] As the immunogen, Cells which are transfected with the
expression vectors pEF-TRAIL-Rldelta and pEF-TRAIL-R2delta, which
contain a DNA encoding human TRAIL-R1 and R2 deleting the death
domain and the amino acids on the C-terminal side from the death
domain in the intracellular region (hereinafter referred to as
"TRAIL-R1 and R2delta"), into L929 cells and excessively express
TRAIL-R1 and R2delta on the cell surfaces are effective.
pEF-TRAIL-R1delta and pEF-TRAIL-R2delta can be prepared by
respectively integrating a DNA encoding a human TRAIL-R1delta
protein and a DNA encoding a human TRAIL-R2delta protein into
pEFneo, expression vectors for animal cells [see Ohashi. H., et al.
(1994) Proc. Natl. Acad. Sci. 91, 158-162]. The DNAs encoding
TRAIL-R1 and R2, vector, host and the like are not limited
thereto.
[0093] Specifically, the transfectant strain obtained by
transfecting L929 cells with pEF-TRAIL-R1 and R2delta is cultured.
Using as indicators the neomycin resistance trait acquired by the
cells having pEFneo vectors inserted therein and the confirmation
of the expression of TRAIL-R1 and R2delta using goat anti-TRAIL-R1
and R2 polyclonal antibodies (DAKO), L929 cells excessively
expressing human TRAIL-R1 and R2delta on the cell surfaces can be
prepared.
[0094] (2) Preparation Step of Antibody-producing Cell
[0095] The antigen obtained in (1), Freund's complete or incomplete
adjuvant or an assistant such as potassium aluminum sulfate are
mixed, and then experimental animals are immunized with the mixture
as an immunogen. Transgenic mice capable of producing human-derived
antibodies are most preferably used as experimental animals, and
such mice are described in the publication of Tomizuka et al
[Tomizuka. et al., Proc Natl Acad Sci USA., 2000 Vol 97: 722].
[0096] The method for administering immunogens upon mouse
immunization may be any of subcutaneous injection, intraperitoneal
injection, intravenous injection, intracutaneous injection,
intramuscular injection or footpad injection. Subcutaneous
injection, intraperitoneal injection, footpad injection or
intravenous injection is preferred.
[0097] Immunization can be performed once, or repeatedly (multiple
times) at appropriate intervals (intervals of preferably 3 days to
1 week or intervals of 2 weeks). Subsequently, the antibody titer
against the antigen in the serum of the immunized animal is
measured, and the animals showing sufficiently increased antibody
titers are used as a source of antibody-producing cells, so that
the effect of the following steps can be enhanced. Generally,
antibody-producing cells derived from animals 3 to 5 days after the
final immunization are preferably used for the following cell
fusion step.
[0098] Examples of the method for measuring antibody titer that is
used herein include various known techniques such as the
radioimmunoassay (hereinafter referred to as "RIA method"),
enzyme-linked immunosorbent assay (hereinafter, referred to as
"ELISA method"), fluorescent antibody method and passive
haemagglutination method. In view of, for example, detection
sensitivity, promptness, correctness, and possibility of automation
of the operation, the RIA method or the ELISA method is more
preferred.
[0099] In the present invention, antibody titer can be measured by
the following procedures according to, for example, the ELISA
method. First, purified or partially purified recombinant human
TRAIL-R1 and R2 are adsorbed on the surface of a solid phase such
as a 96-well plate for ELISA. The solid phase surface, on which no
antigen is adsorbed, is further coated with a protein, which is
independent of the antigen, such as bovine serum albumin
(hereinafter referred to as "BSA"). After the surface is washed, it
is allowed to come into contact with a sample (e.g., mouse serum)
that has been subjected to serial dilution as a primary antibody.
Anti-TRAIL-R1 and R2 antibodies in the sample are bound to the
above antigen. As a secondary antibody, enzyme-labeled antibodies
against human antibodies are added and bound to the human
antibodies. After washing, the substrate of the enzyme is added,
and then changes and the like in absorbance due to color
development resulting from substrate degradation are measured. By
this method, antibody titer is calculated.
[0100] (3) Preparation Step of Myeloma
[0101] As myeloma, cells incapable of producing autoantibodies and
derived from mammals such as mice, rats, guinea pigs, hamsters,
rabbits or humans can be used. In general, established cell lines
obtained from mice, for example, 8-azaguanine-resistant mouse
(derived from BALB/c) myeloma strains P3X63Ag8U.1 (P3-U1) [Yelton,
D. E. et al. Current Topics in Microbiology and Immunology, 81, 1-7
(1978)], P3/NSI/1-Ag4-1(NS-1) [Kohler, G. et al. European J.
Immunology, 6, 511-519 (1976)], Sp2/O-Ag14(SP-2) [Shulman, M. et
al. Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, J.
F. et al. J. Immunology, 123, 1548-1550 (1979)] and P3X63Ag8 (X63)
[Horibata, K. and Harris, A. W. Nature, 256, 495-497 (1975)] are
preferably used. These cell lines are sub-cultured in, for example,
a 8-azaguanine medium [the medium prepared by adding 8-azaguanine
to an RPMI-1640 medium supplemented with glutamine,
2-mercaptoethanol, gentamicin and fetal calf serum (hereinafter
referred to as "FCS")], Iscove's Modified Dulbecco's Medium
(hereinafter referred to as "IMDM") or Dulbecco's Modified Eagle
Medium (hereinafter referred to as "DMEM"). Subculture is performed
using a normal medium 3 to 4 days before cell fusion (e.g., DMEM
medium containing 10% FCS), and 2.times.10.sup.7 or more cells are
ensured at the day of cell fusion.
[0102] (4) Cell Fusion
[0103] Antibody-producing cells are plasma cells, or lymphocytes
that are progenitor cells thereof, and may be obtained from any
site of an individual. In general, the cells can be obtained from,
for example, the spleen, lymph node, bone marrow, tonsil,
peripheral blood or an appropriate combination thereof. Splenocytes
are most generally used.
[0104] After the final immunization, for example, the spleen, which
is a site where antibody-producing cells are present, is excised
from the mouse from which a given antibody titer is obtained,
thereby preparing splenocytes, the antibody-producing cells.
Currently, the most generally employed means for fusing the
splenocytes with the myeloma obtained in step (3) is a method using
polyethylene glycol, which has a relatively low cytotoxicity and
with which the fusion procedure is simple. For example, this method
comprises the following steps.
[0105] Splenocytes and myeloma are washed well in a serum-free
medium (e.g., DMEM) or a phosphate-buffered saline (hereinafter
referred to as "PBS"), and then mixed well to have a cell number
ratio of splenocytes to myeloma of approximately 5:1 to 10:1,
followed by centrifugation. The supernatant is removed, and then
the precipitated cell groups are well disassembled. 1 ml of a 50%
(w/v) polyethylene glycol (molecular weight of 1000 to
4000)-containing serum-free medium is dropped onto the precipitate
while stirring. Subsequently, 10 ml of a serum-free medium is
slowly added, and then centrifugation is performed. The supernatant
is discarded again. The precipitated cells are suspended in a
normal medium containing an appropriate amount of hypoxanthine,
aminopterin, thymidine (hereinafter referred to as "HAT") solution
(hereinafter referred to as "HAT medium") and human interleukin-6
(hereinafter referred to as "IL-6"), added in each well of a plate
for culturing (hereinafter referred to as "plate"), and then
cultured in the presence of 5% carbon dioxide gas at 37.degree. C.
for approximately 2 weeks. Supplementation with a HAT medium is
appropriately performed during culturing.
[0106] (5) Selection of Hybridoma Group
[0107] When the above myeloma cells are cells of an 8-azaguanine
resistant strain, that is, the cells of a hypoxanthine guanine
phosphoribosyltransferase (HGPRT)-deficient strain, unfused myeloma
cells and myeloma-myeloma fusion cells are unable to survive in a
HAT-containing medium. While a fusion cell of two
antibody-producing cells, or a hybridoma of an antibody-producing
cell and a myeloma cell can survive, the fusion cell of two
antibody-producing cells has a limited life span. Thus, when
culturing in a HAT-containing medium is continued, only hybridomas
of antibody-producing cells and myeloma cells survive, so that the
hybridoma can be selected.
[0108] For hybridomas grown to form colonies, the HAT medium is
exchanged with a medium from which aminopterin has been removed
(hereinafter referred to as "HT medium"). Subsequently, a part of
the culture supernatant is collected, and then, for example,
anti-TRAIL-R1 and R2 antibody titers are measured by the ELISA
method. However, when the above fusion protein is used as an
antigen for ELISA, a step of removing clones producing antibodies
that specifically bind to the Fc region of human IgG is required so
as not to select such a clone. The presence or absence of such a
clone can be confirmed by, for example, ELISA using the Fc region
of human IgG as an antigen.
[0109] The method using the 8-azaguanine resistant cell strain is
as illustrated above. Other cell strains can also be used depending
on a selection method for hybridomas. In this case, a medium
composition to be used varies depending on the method used.
[0110] (6) Cloning Step
[0111] Hybridomas that have been shown to produce specific
antibodies by measuring antibody titer in a manner similar to that
described in (2) are transferred to another plate and then
subjected to cloning. Examples of the cloning method include the
limiting dilution method wherein dilution is performed to cause
each well of a plate to contain one hybridoma, followed by
culturing; the soft agar method, wherein culturing is performed in
a soft agar medium and then colonies are collected; a method
wherein each cell is picked with a micromanipulator and then the
cell is cultured; and the sorter clone method, wherein one cell is
separated with a cell sorter. The limiting dilution method is
convenient, and is often used.
[0112] For the wells in which antibody titer has been detected, for
example, cloning is repeated 2 to 4 times by the limiting dilution
method, and then strains that have stable antibody titers are
selected as anti-TRAIL-R1 and R2 monoclonal antibody-producing
hybridoma strains.
[0113] In addition, a mouse-mouse hybridoma H-48-2 which is the
human anti-TRAIL-R2 monoclonal antibody-producing cell of the
present invention, was internationally deposited with International
Patent Organism Depositary at the National Institute of Advanced
Industrial Science and Technology (Central 6, 1-1-1, Higashi,
Tsukuba, Ibaraki, Japan) on May 18, 2001. The international
accession number is FERM BP-7599. In addition, a hybridoma E-11-13
was internationally deposited under the accession number of FERM
BP-7698, a hybridoma F-4-8 under the accession number of FERM
BP-7699, and a hybridoma L-30-10 under the accession number of FERM
BP-7700 on Aug. 8, 2001. In addition, a hybridoma 0304 was
internationally deposited under the accession number of FERM
BP-8037, and a hybridoma KMTR1 under the accession number of FERM
BP-8038 on May 10, 2002. Hence, for example, when antibodies are
prepared using the mouse-mouse hybridomas, the antibodies can be
prepared by step (7) and the following steps (described below)
while omitting steps (1) to (6). Moreover, culturing is performed
in vivo, for example, in mouse ascites, and then antibodies can be
isolated from the ascites.
[0114] (7) Preparation of Monoclonal Antibody by Culturing
Hybridoma
[0115] After the completion of cloning, the hybridoma is cultured
in a normal medium to which HT medium is exchanged.
[0116] Mass culture is performed by the roll-streak system using a
large culture bottle, or by the spinner culture method. The
supernatant in the mass culture is purified using a method known by
a person skilled in the art such as gel filtration, so that
anti-TRAIL-R1 and R2 monoclonal antibodies which are contained in
the prophylactic or the therapeutic agent of the present invention
as an active ingredient can be obtained. Furthermore, proliferation
of the hybridoma intraperitoneally in, for example, mice of the
same line (e.g., BALB/c) or Nu/Nu mice, rats, guinea pigs, hamsters
or rabbits makes it possible to obtain ascites containing a large
amount of anti-TRAIL-R1 and R2 monoclonal antibodies which are
contained in the prophylactic or the therapeutic agent of the
present invention as an active ingredient. As a convenient
purification method, for example, a commercially available
monoclonal antibody purification kit (e.g., MAbTrap GII kit;
Amersham Pharmacia Biotech) can also be used.
[0117] Monoclonal antibodies thus obtained have high antigen
specificity against the human TRAIL-R1 and R2.
[0118] (8) Verification of Monoclonal Antibody
[0119] The isotype and the subclass of the thus-obtained monoclonal
antibody can be determined as follows. Examples of identification
method include the Ouchterlony method, the ELISA method and the RIA
method. Although the Ouchterlony method is convenient, an
enrichment step is required when the concentration of monoclonal
antibodies is low.
[0120] In contrast, when the ELISA method or the RIA method is
used, the culture supernatant is allowed to react intact with an
antigen-coated solid phase. By further using antibodies to various
immunoglobulin isotypes and subclasses as secondary antibodies, the
isotype and the subclass of the monoclonal antibody can be
identified.
[0121] Furthermore, protein quantification can be performed by the
Folin-Lowry method, and a calculation method using absorbance at
280 nm [1.4(OD280)=immunoglobulin 1 mg/ml].
[0122] Epitopes to be recognized by monoclonal antibodies can be
identified as follows. First, various partial structures of a
molecule that the monoclonal antibody recognizes are prepared. To
prepare the partial structures, for example, there exist a method
whereby various partial peptides of the molecule are produced using
a known oligopeptide synthesis technique and a method whereby DNA
sequences encoding target partial peptides are integrated into
appropriate expression plasmids using genetic engineering
techniques, and then the peptides are produced inside and outside a
host such as Escherichia coli. In general, both methods are used in
combination for the above purpose. For example, a series of
polypeptides are prepared to be appropriately shorter in length
sequentially from the C-terminus or the N-terminus of an antigen
protein, using a genetic engineering technique known to a person
skilled in the art. Then, the reactivities of the monoclonal
antibody against them are studied, so that the approximate
recognition site is determined.
[0123] Next, more specifically, various oligopeptides corresponding
to the site, mutants or the like of the peptides are synthesized
using an oligopeptide synthesis technique known to a person skilled
in the art. Then, the ability of the monoclonal antibody (contained
as an active ingredient in the prophylactic or the therapeutic
agent of the present invention) to bind to these peptides is
examined, or the activity of competitive inhibition of the peptide
on the binding of the monoclonal antibody with the antigen is
examined, thereby specifying the epitope. As a convenient method
for obtaining various oligopeptides, a commercially available kit
(e.g., SPOTs kit, GENOSYS BIOTECHNOLOGIES), a kit for a series of
multipin peptide synthesis (Chiron) using the multipin syntheses
method or the like can also be used.
[0124] Moreover, a gene encoding a human monoclonal antibody is
cloned from an antibody-producing cell such as a hybridoma, the
gene is integrated into an appropriate vector, and then the vector
is introduced into a host (e.g., a mammalian cell line, Escherichia
coli, yeast cells, insect cells or plant cells). Thus, recombinant
antibodies that are produced using the gene recombinant technique
can be prepared (P. J. Delves., ANTIBODY PRODUCTION ESSENTIAL
TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean., Monoclonal
Antibodies., 2000 OXFORD UNIVERSITY PRESS, J. W. Goding.,
Monoclonal Antibodies: principles and practice., 1993 ACADEMIC
PRESS).
[0125] A method employed to prepare a gene encoding a monoclonal
antibody from a hybridoma comprises the step of preparing by the
PCR method and the like DNAs respectively encoding the light chain
variable region, light chain constant region, heavy chain variable
region and heavy chain constant region of the monoclonal antibody.
In this case, oligo DNAs designed from the anti-TRAIL-R antibody
gene or amino acid sequence can be used as primers, and DNA
prepared from the hybridoma can be used as a template. These DNAs
are integrated into an appropriate vector, and then the vector is
introduced into a host for expression. Alternatively, these DNAs
are separately integrated into appropriate vectors, thereby causing
co-expression.
[0126] Examples of vectors used herein include phages or plasmids
that can autonomously grow in a host microorganism. Examples of the
plasmid DNA include plasmids derived from Escherichia coli,
Bacillus subtilis or yeast. An example of the phage DNA is a X
phage.
[0127] Examples of the host used for transformation are not
specifically limited, as long as it can express a target gene, and
include bacteria (e.g., Escherichia coli and Bacillus subtilis),
yeast, animal cells (e.g., COS cells and CHO cells) and insect
cells.
[0128] Methods for introducing a gene into a host are known, and
any such method may be used (e.g., a method using calcium ion, the
electroporation method, the spheroplast method, the lithium acetate
method, the calcium phosphate method and the lipofection method).
In addition, examples of a method for introducing a gene into an
animal (described later) include the microinjection method, a
method for introducing a gene into ES cells by the electroporation
or the lipofection method, and the nucleus transplantation
method.
[0129] In the present invention, anti-TRAIL-R antibodies can be
obtained by culturing transformants and collecting the antibodies
from the culture product. The term "culture product" means any of
(a) a culture supernatant, (b) cultured cells or cultured microbes
or the disrupted cells or microbes thereof, or (c) the secretion
product of the transformant. To culture transformants, a medium
appropriate for the host used herein is used, and the static
culture method, a culture method using a roller bottle or the like
is employed.
[0130] After culturing, when a target protein is produced within
microbes or cells, antibodies are collected by disrupting the
microbes or the cells. Furthermore, when a target antibody is
produced outside the microbes or the cells, the culture solution is
used intact, or the microbes or cells are removed by centrifugation
or the like. Subsequently, a target antibody can be isolated and
purified from the above culture product using one of or an
appropriate combination of general biochemical methods with various
chromatographies that are used for protein isolation and
purification. If necessary, a monomer of an antibody can be
separated and purified by a method such as gel filtration.
[0131] Moreover, by the use of transgenic animal generation
techniques, animal hosts having the gene of a target antibody
integrated in the endogenous gene are generated, such as transgenic
cattle, transgenic goats, transgenic sheep or transgenic pigs. The
antibody gene-derived monoclonal antibodies can then be obtained in
large quantities from the milk to be secreted from the transgenic
animal (Wright, G., et al. (1991) Bio/Technology 9, 830-834).
Hybridomas can be cultured in vitro using a known nutrition medium,
which is used to allow the proliferation, maintenance, and storage
of the hybridoma so as to cause the hybridoma to produce monoclonal
antibodies in the culture supernatant depending on various
conditions such as the characteristics of a cell type to be
cultured, the purpose of an experiment or study, and a culture
method; or any nutrition medium, which is induced and prepared from
a known basic medium.
[0132] (9) Characteristics of Antibody
[0133] The antibody of the present invention has the following
functional properties (a) to (c), and each of the properties can be
confirmed by, for example, the method described for each of (a) to
(c).
[0134] (a) When human carcinoma cells are cultured, the antibody of
the present invention is contained in the medium, and the survival
rate of the cells is examined, the antibody has activity to induce
apoptosis in carcinoma cells expressing TRAIL-R1 and/or R2.
[0135] (b) When normal human tissue-derived cells are cultured, the
antibody of the present invention is contained in the medium, and
the survival rate of the cells is examined, the antibody does not
have effect on normal cells expressing TRAIL-R1 and/or R2.
[0136] (c) When human hepatocytes are cultured, the antibody of the
present invention is contained in the medium, and the survival rate
of the cells is examined, the antibody does not induce hepatocyte
toxicity.
[0137] The apoptosis-inducing activity of the antibody of the
present invention can be expressed using an LD50 value (an antibody
concentration, which causes death in half of the cells under a
given experimental condition) as an indicator. The LD50 value is
100 .mu.g/ml or less, preferably 10 .mu.g/ml or less, more
preferably 0.7 .mu.g/ml or less, further preferably 0.02 to 0.11
.mu.g/ml, or most preferably 0.02 .mu.g/ml or less in the
experimental conditions described hereinafter.
[0138] Furthermore, the term "does not have effect on normal cells"
or "does not induce hepatocyte toxicity" means that the
apoptosis-inducing activity of the antibody of the present
invention on normal cells (human hepatocytes) is not significantly
high. When an LD50 value is used as an indicator, it is 0.01
.mu.g/ml or more, preferably 0.1 .mu.g/ml or more, more preferably
2 to 10 .mu.g/ml, further preferably 10 to 24 .mu.g/ml or most
preferably 24 .mu.g/ml or more in the experimental conditions
described hereinafter.
[0139] The antibody of the present invention has any of the above
activities (a) to (c). The antibody is a substance having novel
characteristics in that it preferably has the above activity (a) of
inducing apoptosis in carcinoma cells, and the above activities (b)
and (c) of not inducing damage on normal cells, particularly normal
hepatocytes. Therefore, the antibody of the present invention is
useful as an ingredient to be contained in a prophylactic or
therapeutic agent against malignant tumors.
[0140] Apoptosis-inducing activity on normal cells or carcinoma
cells can be expressed using an LD50 value as an indicator. The
LD50 value in the present invention can be calculated as follows.
Normal cells (e.g., human heptocytes) or carcinoma cells (e.g.,
human colon cancer cell line Colo205; ATCC CCL-222) are cultured,
and then the antibody of the present invention is added to a
medium. After a certain period of time, the survival rate of the
cells is measured by MTT assay (Green, L. M. et al., J.
Immunological Methods, 70: 257-268 (1984)), LDH assay or the
like.
[0141] Based on a graph on which the survival rate and the
concentration of the antibody added are plotted, an antibody
concentration corresponding to a survival rate of 50% is determined
as an LD50 value.
[0142] The LD50 value can be read from a graph, or calculated by
finding a formula for a graph curve by regression calculation.
[0143] In an experiment for carcinoma cells (Colo205),
2.5.times.10.sup.3 cells are seeded in 100 .mu.l of a medium per
well of a 96-well flat-bottomed plate, and then cultured at
37.degree. C. in the presence of 5% CO.sub.2. On the next day, the
antibodies are added, the mixture is allowed to stand for 48 hours
in the above environment, and then the survival rate of the cells
is measured (total volume of the reaction solution: 110 to 120
.mu.l). In the present invention, the above conditions are
described as "number of cells: 2.5.times.10.sup.3 and reaction
time: 48 hours."
[0144] In an experiment for normal cells (hepatocytes),
7.5.times.10.sup.4 cells are seeded in 100 .mu.l of a medium per
well of a 96-well flat-bottomed plate, and then cultured at
37.degree. C. in the presence of 5% CO.sub.2. On the next day, the
antibodies are added, the mixture is allowed to stand for 24 hours
in the above environment, and then the survival rate of the cells
is measured (total volume of the reaction solution: 110 to 120
.mu.l). In the present invention, the above conditions are
described as "number of cells: 7.5.times.104 and reaction time: 24
hours."
[0145] The antibody of the present invention includes antibodies
having a property of showing an LD50 value for normal cells (human
hepatocytes) of, for example, 0.01 .mu.g/ml (10 ng/ml) or more, or
preferably 0.1 .mu.g/ml or more when the LD50 value is measured
under the above conditions. It can be said that the higher the LD50
value against normal cells, the higher the safety. Thus, antibodies
having LD50 values of 2 to 100 .mu.g/ml are further preferred. The
antibody of the present invention includes antibodies having a
property of showing the LD50 value for carcinoma cells of, for
example, 100 .mu.g/ml or less, or preferably 0.7 .mu.g/ml or less
when the LD50 value is measured under the above conditions. It can
be said that lower the LD50 value against carcinoma cells, the
stronger the activity to kill tumor cells. Thus, antibodies having
LD50 values of 0.02 to 0.11 .mu.g/ml are further preferred. In
particular, the E-11-13 antibody, the L-30-10 antibody and the
KMTR1 antibody of the present invention have properties of showing
LD50 values for human hepatocytes of 2 to 100 .mu.g/ml or more (for
example, 2 to 24 .mu.g/ml, preferably, 100 .mu.g/ml) and LD50
values for carcinoma cells of 0.02 to 0.11 .mu.g/ml. That is, these
antibodies have both safety for normal cells and an
apoptosis-inducing effect on tumor cells. Furthermore surprisingly,
the antibody of the present invention significantly suppressed
tumor cell proliferation in a tumor-bearing mouse model.
[0146] The ratio of the LD50 value for normal cells measured under
conditions where "number of cells: 7.5.times.10.sup.4 and reaction
time: 24 hours" to the LD50 value for carcinoma cells measured
under conditions where "number of cells: 2.5.times.10.sup.3 and
reaction time: 48 hours" is next examined. As described above, the
higher the LD50 value for normal cells, the higher the safety, and
the lower the LD50 value for carcinoma cells, the stronger the
activity to kill tumor cells. Hence, antibodies having a higher
ratio of the LD50 value for normal cells to that for carcinoma
cells can be said to be useful (higher safety and stronger
apoptosis-inducing activity in carcinoma cells). The ratio of the
LD50 value for carcinoma cells to that for normal cells (showing
how many times the LD50 value for normal cells is greater than that
for carcinoma cells) is supposed to be an N/C ratio. The antibody
of the present invention has a property of having N/C=2 or more,
namely, having a LD50 value for normal cells which is twice or more
greater than that for carcinoma cells. Preferably the LD50 for
normal cells is 10 times or more greater (N/C=10 or more) than that
for carcinoma cells, more preferably, N/C=10 to 25. Hereinafter,
the N/C ratio, in order of preference, is as follows: N/C=50,
N/C=50 or more, N/C=50 to 100, N/C=100 or more, N/C=100 to 1000,
N/C=250 to 1000 and most preferably N/C=1000 or more.
[0147] Furthermore, when a monomer antibody of the present
invention is added to culture medium and human carcinoma cells are
cultured in the medium, the antibody has the activity to induce
apoptosis to carcinoma cells expressing TRAIL-R1 and/or TRAIL-R2
independently of exogenous factors.
[0148] Pharmaceutical Composition
[0149] A preparation containing a preparation that is prepared by
purifying the human anti-TRAIL-R1 and R2 antibodies of the present
invention is also encompassed within the scope of the present
invention. Such a preparation preferably contains a physiologically
acceptable diluent or carrier in addition to the antibody, and may
be a mixture with other antibodies or other drugs such as
antibiotics. Examples of an appropriate carrier include, but are
not limited to, a physiological saline solution, a phosphate
buffered saline solution, a phosphate buffered saline glucose
solution and a buffered physiological saline. Alternatively, the
antibody may be freeze-dried, and then used when necessary by
adding the above buffered aqueous solution for reconstitution. The
prophylactic or therapeutic agent can be administered in various
forms. Examples of the forms of administration of these agents
include oral administration using vehicles such as tablets,
capsules, granules, powders or syrups, and parenteral
administration using vehicles such as injections, drops or
suppositories.
[0150] The dose differs depending on symptom, age, body weight and
the like. Normally in the case of oral administration, the dose is
approximately 0.01 mg to 1000 mg per day for an adult, and it can
be administered once or separately administered on several
different occasions. Further, in the case of parenteral
administration, a dose of approximately 0.01 mg to 1000 mg per
administration can be administered by subcutaneous injection,
intramuscular injection or intravenous injection.
[0151] The antibody or the pharmaceutical composition of the
present invention can be applied to treatment of or prophylaxis
against various diseases or symptoms that may be caused by cells
expressing TRAIL-R1 and R2. Examples of such diseases or the
symptoms include various malignant tumors.
[0152] Examples of the types of such tumors include colon cancer,
colorectal cancer, lung cancer, breast cancer, brain tumor,
malignant melanoma, renal cell carcinoma, bladder cancer, leukemia,
lymphomas, T cell lymphomas, multiple myeloma, gastric cancer,
pancreas cancer, cervical cancer, endometrial carcinoma, ovarian
cancer, esophageal cancer, liver cancer, head and neck squamous
cell carcinoma, cutaneous cancer, urinary tract carcinoma, prostate
cancer, choriocarcinoma, pharyngeal cancer, laryngeal cancer,
thecomatosis, androblastoma, endometrium hyperplasy, endometriosis,
embryoma, fibrosarcoma, Kaposi's sarcoma, hemangioma, cavernous
hemangioma, angioblastoma, retinoblastoma, astrocytoma,
neurofibroma, oligodendroglioma, medulloblastoma,
ganglioneuroblastoma, glioma, rhabdomyosarcoma, hamartoblastoma,
osteogenic sarcoma, leiomyosarcoma, thyroid sarcoma and Wilms
tumor. The number of the types of tumors to which the antibody of
the present invention is applied is not limited to one type, and
plural types of tumors may develop at the same time.
Example of Preparation
[0153] The molecule of the present invention is used in the form of
an ampule of aseptic solution or suspension prepared by dissolving
the molecule in water or a pharmacologically acceptable solution
other than water. In addition, an ampule may be filled with an
aseptic powder preparation (preferably, where the molecule of the
present invention is freeze-dried), and it can be diluted with a
pharmacologically acceptable solution when used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0154] FIG. 1 shows the cell-death-inducing activity on Colo205 in
the culture supernatant of hybridomas producing human anti-TRAIL-R1
monoclonal antibodies.
[0155] FIG. 2 shows the cell-death-inducing activity on Colo205 in
the culture supernatant of hybridomas producing human anti-TRAIL-R2
monoclonal antibodies.
[0156] FIG. 3 shows the cell-death-inducing activity on Colo205 in
the culture supernatant of hybridomas producing human anti-TRAIL-R2
monoclonal antibodies (Goat anti-human IgG(.gamma.) specific
polyclonal antibodies were not added).
[0157] FIG. 4 shows the cell-death-inducing activity on HUVEC in
the culture supernatant of hybridomas producing human anti-TRAIL-R2
monoclonal antibodies.
[0158] FIG. 5a shows the cell-death-inducing activity of human
recombinant TRAIL (positive control) on Colo205 and normal human
hepatocytes.
[0159] FIG. 5b shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (H-48-2) on Colo205 and
normal human hepatocytes.
[0160] FIG. 5c shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (E-11-13) on Colo205 and
normal human hepatocytes.
[0161] FIG. 5d shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (L-30-10) on Colo205 and
normal human hepatocytes.
[0162] FIG. 5e shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (F-4-8) on Colo205 and
normal human hepatocytes.
[0163] FIG. 5f shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (W-40-5) on Colo205 and
normal human hepatocytes.
[0164] FIG. 5g shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (0304) on Colo205 and
normal human hepatocytes.
[0165] FIG. 5h shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (0322) on Colo205 and
normal human hepatocytes.
[0166] FIG. 5i shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (KMTR1) on Colo205 and
normal human hepatocytes.
[0167] FIG. 5j shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (DIM) on Colo205 and
normal human hepatocytes.
[0168] FIG. 5k shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (0304, Goat anti-human
IgG antibodies were not added) on Colo205 and normal human
hepatocytes.
[0169] FIG. 5l shows the cell-death-inducing activity of purified
human anti-TRAIL-R2 monoclonal antibodies (KMTR1, Goat anti-human
IgG antibodies were not added) on Colo205 and normal human
hepatocytes.
[0170] FIG. 6 shows the results of measuring the tumor size when
purified human anti-TRAIL-R2 monoclonal antibodies E-11-13, F-4-8,
H-48-2, L-30-10 and W-40-5 were administered at 1 .mu.g/mouse three
times on alternate days.
[0171] FIG. 7 shows the results of measuring the tumor size when
purified human anti-TRAIL-R2 monoclonal antibodies E-11-13 were
administered at 4, 20 and 100 .mu.g/mouse 4 times.
[0172] FIG. 8 shows the results of measuring the tumor size when
purified human anti-TRAIL-R2 monoclonal antibodies E-11-13 were
administered at 20 .mu.g/300 mm.sup.3 tumor-bearing mouse three
times on alternate days.
[0173] FIG. 9 shows the results of measuring the tumor size when
purified human anti-TRAIL-R2 monoclonal antibodies 0304 were
administered at 20 .mu.g/100 mm.sup.3 tumor-bearing mouse three
times on alternate days.
[0174] FIG. 10 shows the results of measuring the tumor size when
purified human anti-TRAIL-R2 monoclonal antibodies 0304 were
administered at 25 .mu.g/100 mm.sup.3 tumor-bearing mouse three
times.
[0175] FIG. 11a shows the cell-death-inducing activity (Goat
anti-human IgG antibodies were not added) of recombinant purified
human anti-TRAIL-R2 monoclonal antibodies on Colo205 cells.
[0176] FIG. 11b shows the cell-death-inducing activity (Goat
anti-human IgG antibodies were added) of recombinant purified human
anti-TRAIL-R2 monoclonal antibodies on Colo205 cells.
[0177] FIG. 12a shows the cell-death inducing activity (Goat
anti-human IgG antibodies were not added, without a cross-linker)
of purified human anti-TRAIL-R2 monoclonal antibodies (0304 and
0322) and control antibody on Colo205 cells.
[0178] FIG. 12b shows the cell-death inducing activity (Goat
anti-human IgG antibodies were added, with a cross-linker) of
purified human anti-TRAIL-R2 monoclonal antibodies (0304 and 0322)
and control antibody on Colo205 cells.
[0179] FIG. 13 shows the antigen biding activity of recombinant
purified human anti-TRAIL-R2 monoclonal antibodies 0304-IgG1 and
0304-IgG4.
[0180] FIG. 14a shows the cell-death inducing activity (Goat
anti-human IgG antibodies were not added, without a cross-linker)
of recombinant purified human anti-TRAIL-R2 monoclonal antibodies
0304-IgG1 and 0304-IgG4 on Colo205 cells.
[0181] FIG. 14b shows the cell-death inducing activity (Goat
anti-human IgG antibodies were added, with a cross-linker) of
recombinant purified human anti-TRAIL-R2 monoclonal antibodies
0304-IgG1 and 0304-IgG4 on Colo205 cells.
[0182] FIG. 15a shows the results of measuring the tumor size when
purified recombinant human anti-TRAIL-R2 monoclonal antibodies
0304-IgG1 were administered at 4, 20 or 100 .mu.g/100 mm.sup.3
tumor-bearing mouse three times.
[0183] FIG. 15b shows the results of measuring the tumor size when
purified recombinant human anti-TRAIL-R2 monoclonal antibodies
0304-IgG4 were administered at 4, 20 or 100 .mu.g/100 mm.sup.3
tumor-bearing mouse three times.
[0184] FIG. 16a shows the results of the fractionation of purified
human anti-TRAIL-R2 monoclonal antibodies (0304) by gel
filtration.
[0185] FIG. 16b shows the results of the fractionation of purified
human anti-TRAIL-R2 monoclonal antibodies (0322) by gel
filtration.
[0186] FIG. 16c shows the results of the fractionation of purified
human anti-TRAIL-R2 monoclonal antibodies (H-48-2) by gel
filtration.
[0187] FIG. 17a shows the cell-death inducing activity (Goat
anti-human IgG antibodies were not added, without a cross-linker)
of each fraction of human anti-TRAIL-R2 monoclonal antibodies
(0304) collected by gel filtration purification on Colo205
cells.
[0188] FIG. 17b shows the cell-death inducing activity (Goat
anti-human IgG antibodies were added, with a cross-linker) of each
fraction of human anti-TRAIL-R2 monoclonal antibodies (0304)
collected by gel filtration purification on Colo205 cells.
[0189] FIG. 17c shows the cell-death inducing activity (Goat
anti-human IgG antibodies were not added, without a cross-linker)
of each fraction of human anti-TRAIL-R2 monoclonal antibodies
(0322) collected by gel filtration purification on Colo205
cells.
[0190] FIG. 17d shows the cell-death inducing activity (Goat
anti-human IgG antibodies were added, with a cross-linker) of each
fraction of human anti-TRAIL-R2 monoclonal antibodies (0322)
collected by gel filtration purification on Colo205 cells.
[0191] FIG. 17e shows the cell-death inducing activity (Goat
anti-human IgG antibodies were not added, without a cross-linker)
of each fraction of human anti-TRAIL-R2 monoclonal antibodies
(H-48-2) collected by gel filtration purification on Colo205
cells.
[0192] FIG. 17f shows the cell-death inducing activity (Goat
anti-human IgG antibodies were added, with a cross-linker) of each
fraction of human anti-TRAIL-R2 monoclonal antibodies (H-48-2)
collected by gel filtration purification on Colo205 cells.
[0193] FIG. 18a shows the relationship between the cell-death
inducing activity on Colo205 cells and a concentration of a
competition antibody (aHSA) when a monomer fraction of human
anti-TRAIL-R2 monoclonal antibodies (0304-IgG1 and H-48-2)
collected by gel filtration purification was added and then goat
anti-human IgG antibody and the competition antibody were added to
be co-existed.
[0194] FIG. 18b shows the relationship between the cell-death
inducing activity on Colo205 cells and a concentration of a
competition antibody (aHSA) when a monomer fraction of human
anti-TRAIL-R2 monoclonal antibodies (0304-IgG1 and H-48-2)
collected by gel filtration purification was added and then human
peripheral blood mononuclear cells (hPBMC) and the competition
antibody were added to be co-existed.
[0195] FIG. 19a shows the relationship between the cell-death
inducing activity on Colo205 cells and Fc receptor when a monomer
fraction of human anti-TRAIL-R2 monoclonal antibodies (0304-IgG1
and H-48-2) collected by gel filtration purification, human
peripheral blood mononuclear cells (hPBMC) and a competition
antibody were co-existed.
[0196] FIG. 19b shows the relationship between the cell-death
inducing activity on Colo205 cells and Fc receptor when a monomer
fraction of human anti-TRAIL-R2 monoclonal antibodies (0304-IgG1
and H-48-2) collected by gel filtration purification, human
peripheral blood mononuclear cells (hPBMC) and a FcR blocker were
co-existed.
[0197] FIG. 20 shows the results of the analysis of the molecular
weight distribution of a complex formed by a monomer fraction of
human anti-TRAIL-R2 monoclonal antibodies (0304-IgG1 and H-48-2)
collected by gel filtration purification and TRAIL-R2 molecules on
the surface of Colo205 cells.
BEST MODE FOR CARRYING OUT THE INVENTION
[0198] The present invention will be described more specifically by
the following examples. The present invention is not limited to the
embodiments described in these examples.
EXAMPLE 1
Preparation of Antigen
[0199] To obtain cells excessively expressing human TRAIL-R1 and R2
on the cell membrane, plasmid vectors for the expression of human
TRAIL R1 and human TRAIL-R2 (which had been prepared by removing
the death domain and the amino acids on the C-terminal side from
the death domain in the intracellular regions from the full-length
amino acids of the human TRAIL-R1 and R2, hereinafter referred to
as TRAIL-R1 and R2delta,) were prepared. DNAs encoding TRAIL-R1 and
R2delta were prepared by the PCR method.
[0200] a) Construction of Full-length Human TRAIL-R1 and R2
Expression Vectors
[0201] To perform template PCR, plasmid vectors, pcDNA3-TRAIL-R1
and pcDNA3-TRAIL-R2, retaining cDNAs encoding human TRAIL-R1 and R2
were used as templates. pcDNA3-TRAIL-R1 and pcDNA3-TRAIL-R2 were
constructed by the following method. The full-length human TRAIL-R1
DNA and TRAIL-R2 DNA were modified by polymerase chain reaction
(PCR) to add an EcoR I sequence to the 5' end, and a Not I sequence
and a termination codon to the 3' end. Using human placenta-derived
cDNA (Clontech) as a template, primers
5'-CACGAATTCACCATGGCGCCACCACCAGCT-3' (SEQ ID NO: 1) and
5'-TTTCTCGAGGCGGCCGCTTATCACTCCAAGGACACGGCAGAGCCTGT G-3' (SEQ ID NO:
2) synthesized for TRAIL-R1, and primers
5'-CACGAATTCGCCACCATGGAACAACGGGGACA- G-3' (SEQ ID NO: 3) and 5
'-TTTCTCGAGGC GGCC GCTCATTAGGACATGGCAGAGTCTGCATT- ACCT-3' (SEQ ID
NO: 4) synthesized for TRAIL-R2, a PCR reaction was performed for
30 cycles (each cycle consisting of 94.degree. C. for 20 seconds,
60.degree. C. for 30 seconds and 68.degree. C. for 90 seconds)
using platinum PfxDNA polymerase (Gibco BRL). The modified TRAIL-R1
and TRAIL-R2 sequences were isolated as EcoR I-Not I fragments, and
then ligated to pcDNA3 (Invitrogen) vectors that had been cleaved
with the same enzymes. The obtained plasmids were named
pcDNA3-TRAIL-R1 and pcDNA3-TRAIL-R2. Of the two fragments formed by
alternative splicing, TRAIL-R2 integrated in pcDNA3-TRAIL-R2
comprises 440 amino acids encoded by a 1320 bp cDNA. Hereinafter,
the reaction temperature for all the PCRs in the examples was
regulated using a GeneAmp PCR system 9700 (Perkin Elmer Japan).
[0202] b) Construction of Human TRAIL-R1 and R2Delta Expression
Vectors
[0203] Human TRAIL-R1 and R2delta expression vectors were
constructed by the following methods. To prepare an expression
plasmid comprising a TRAIL-R1 partial peptide having an amino acid
sequence of 1 to 351, and the same comprising a TRAIL-R2 partial
peptide having an amino acid sequence of 1 to 348, PCR reaction was
performed to add an EcoR I sequence to the 5' ends of the TRAIL-R1
and R2 partial peptides, and an Not I sequence and a termination
codon to the 3' ends of the same. PCR was performed for 25 cycles
(each cycle consisting of 94.degree. C. for 20 seconds, 65.degree.
C. for 30 seconds and 68.degree. C. for 75 seconds) using
oligonucleotide primers 5'-CACGAATTCACCATGGCGCCACCACCAGCT-- 3' (SEQ
ID NO: 1) and 5'-TTCTACGAGCGGCTTATCACAGCCTCCTCCTCTGAGA-3' (SEQ ID
NO: 5) for TRAIL-R1, and oligonulcotide primers
5'-CACGAATTCGCCACCATGGAAC- AACGGGGACAG-3' (SEQ ID NO: 3) and
5'-TTCTACGAGCGGCCGCTTATCACAAGTCTGCAAAGTC- ATC-3' (SEQ ID NO: 6) for
TRAIL-R2, .mu.latinum PfxDNA polymerase (Gibco BRL),
pcDNA3-TRAIL-R1 and pcDNA3-TRAIL-R2. The modified TRAIL-R1 and R2
partial peptides were isolated as EcoR I-Not I fragments. The EcoR
I-Not I fragment was ligated to pEFneo vectors that had been
cleaved with EcoR I and Not I enzymes. The obtained plasmids were
named pEF-TRAIL-R1delta and pEF-TRAIL-R2delta.
[0204] c) Preparation of Human TRAIL-R1 and R2Delta-expressing
Cells
[0205] pEF-TRAIL-R1delta and pEF-TRAIL-R2delta prepared in b) were
introduced into L929 cells (American Type Culture Collection
No.CCL-1) using LipofectAMINE Plus (Gibco BRL). Transfection was
performed by the method described in the manual. After 24 hours of
culturing in a flask for culturing cells (with a culture area of 75
cm.sup.2) at 37.degree. C. under 5.0% carbon dioxide gas, G418
(Gibco BRL) was added at 1 mg/ml in the culture, followed by 1 week
of culturing. Subsequently, FACS analysis was performed using goat
anti-human TRAIL-R1 polyclonal antibodies and goat anti-human
TRAIL-R2 polyclonal antibodies (DAKO). Thus, it was confirmed that
the transfected cells, which had acquired a G418 resistance trait,
expressed TRAIL-R1delta comprising 351 amino acids and
TRAIL-R2delta comprising 348 amino acids on the cell membrane
surface.
[0206] The synthesis of oligonucleotides such as primers for PCR
was always performed using an automated DNA synthesis system (model
3948, Perkin Elmer Japan, Applied Biosystems division) according to
the manual [see Matteucci, M. D. and Caruthers, M. H. (1981) J. Am.
Chem. Soc. 103, 3185-3191]. After the end of synthesis, each
oligonucleotide was cleaved from the support and then deprotected.
The obtained solution was dried and solidified, and the product was
dissolved in distilled water, and then cryopreserved at -20.degree.
C. until use.
Example 2
Generation of Human Antibody-producing Mice
[0207] The mice used for immunization had a genetic background
whereby they were homozygotes for both disrupted endogenous Ig
heavy chain and .kappa. light chain, and the mice harbored at the
same time chromosome 14 fragment (SC20) containing a human Ig heavy
chain locus, and a human Ig.kappa. chain transgene (KCo5). These
mice were generated by crossing mice of a line A having a human Ig
heavy chain locus with mice of a line B having a human Ig.kappa.
chain transgene. The mice of line A are homozygotes for both
disrupted endogenous Ig heavy chain and .kappa. light chain, and
harbor chromosome 14 fragment (SC20), which is transmittable to
progeny, as is described, for example, in the report of Tomizuka et
al. (Tomizuka. et al., Proc Natl Acad Sci USA., 2000 Vol 97: 722).
Furthermore, the mice of line B (transgenic mice) are homozygotes
for both disrupted endogenous Ig heavy chain and .kappa. light
chain, and harbor a human Ig.kappa. chain transgene (KCo5), as
described, for example, in the report of Fishwild et al. (Nat
Biotechnol., 1996 Vol 14:845).
[0208] Progeny mice obtained by crossing male mice of the line A
with female mice of the line B, or female mice of the line A with
male mice of the line B, were analyzed by the method described in
Tomizuka et al's report (Tomizuka et al., Proc Natl Acad Sci USA.,
2000 Vol 97:722). Individuals (human antibody-producing mice)
having human Ig heavy chain and .kappa. light chain detected
simultaneously in the sera were screened for (Ishida & Lonberg,
IBC's 11th Antibody Engineering, Abstract 2000 ; Ishida, I. et al.,
Cloning & Stem Cells 4, 85-96 (2002)) and used for the
following immunization experiment. Mice having the altered genetic
background of the above mice and the like (Isao ISHIDA, 2002,
JIKKEN IGAKU 20, 6, 846-851) were also used for the immunization
experiment. In addition, the above human antibody-producing mice
are available from Kirin Brewery Co., Ltd via contract.
Example 3
Preparation of Human Monoclonal Antibodies Against Human TRAIL-R1
and R2
[0209] In this example, monoclonal antibodies were prepared
according to general methods as described in, for example,
Introduction of Experimental Protocols for Monoclonal Antibody
(Monoclonal Antibody Jikken Sosa Nyumon, written by Tamie ANDO et
al., KODANSHA, 1991). As immunogens, the TRAIL-R1 and
R2delta-expressing L929 cell prepared in Example 1 or a fusion
protein of the extracellular regions of human TRAIL-R1 and R2 and
the Fc region of human IgG1 was used. Animals used for immunization
were the human antibody (human immunoglobulin)-producing mice
generated in Example 2.
[0210] To prepare human monoclonal antibodies against human
TRAIL-R1, human antibody-producing mice were initially immunized
via the right foot pad with the TRAIL-R1delta-expressing L929 cells
(3.times.10.sup.6 cells/mouse) prepared in Example 1. After the
initial immunization, immunization with the L929 cells was
performed 10 times every 3 days via the left and right food pad
alternatively. Furthermore, at 3 days before the obtainment of the
spleen and the lymph node (described later), the L929 cells were
used for immunization via both foot pads. To prepare human
monoclonal antibodies against human TRAIL-R2, human
antibody-producing mice (1.times.10.sup.7 cells/mouse) were
initially immunized intraperitoneally with the
TRAIL-R2delta-expressing L929 cells prepared in Example 1. After
the initial immunization, immunization with the L929 cells was
performed 5 or 6 times a week intraperitoneally. Furthermore, at 3
days before the obtainment of the spleen (described later) the L929
cells or a fusion protein of the extracellular region of human
TRAIL-R2 and the Fc region of human IgG1 was used for immunization
via the caudal vein. Moreover, the fusion protein comprising the
extracellular region of human TRAIL-R2 and the Fc region of human
IgG1 was mixed with Freund's complete adjuvant. The human
antibody-producing mice were then initially immunized
subcutaneously with the mixture. Subsequently, the same protein was
mixed with Freund's incomplete adjuvant, and the mice were
immunized twice subcutaneously with the mixture every 2 weeks.
Furthermore, at 3 days before the obtainment of the spleen
(described later), the same protein was used for immunization via
the caudal vein.
[0211] The spleens and/or the lymph nodes were obtained by a
surgical operation from the immunized mice. Then the organ was put
into 10 ml of a serum-free DMEM medium (Gibco BRL) containing 350
mg/ml sodium hydrogen carbonate, 50 units/ml penicillin and 50
.mu.g/ml streptomycin (hereinafter, referred to as "serum-free DMEM
medium"). It was then pulverized using a spatula on mesh (Cell
strainer: Falcon). The cell suspension that had passed through the
mesh was centrifuged so as to precipitate the cells. The cells were
washed twice in a serum-free DMEM medium, and suspended in a
serum-free DMEM medium, and then the number of the cells was
counted. In the meantime, myeloma cells SP2/0 (ATCC No. CRL-1581)
that had been cultured so as not to exceed a cell concentration of
1.times.10.sup.8 cells/ml at 37.degree. C. in the presence of 5%
carbon dioxide gas in a 10% FCS (Sigma)-containing DMEM medium
(Gibco BRL) (hereinafter referred to as "serum-containing DMEM
medium") were washed in a serum-free DMEM medium in the same
manner. Then the cells were suspended in a serum-free DMEM medium,
and then the number of the cells was counted. The collected cell
suspension and the mouse myeloma suspension were mixed at a cell
number ratio of 5:1. The mixture was centrifuged, thereby
completely removing the supernatant. To this pellet, 1 ml of 50%
(w/v) polyethylene glycol 1500 (Boehringer Mannheim) was gently
added as a fusion agent while agitating the pellet using the tip of
a pipette. Next, 1 ml of a serum-free DMEM medium preheated at
37.degree. C. was gently added at two separate times, followed by
addition of another 7 ml of serum-free DMEM medium. After
centrifugation, the fusion cells obtained by the removal of the
supernatant were subjected to screening by the limiting dilution
method described below. Screening for hybridomas was performed by
culturing the cells in DMEM media containing 10% fetal calf serum
(FCS), hypoxanthine (H), aminopterin (A) and thymidine (T)
(hereinafter referred to as "HAT": Sigma). Further, single clones
were obtained using DMEM media containing 10% FCS and HT (Sigma) by
the limiting dilution method. Culturing was performed in a 96-well
microtiter plate (Beckton Dickinson). Screening for hybridoma
clones producing anti-human TRAIL-R1 and R2 human monoclonal
antibodies and characterization of the human monoclonal antibodies
produced by each of the hybridomas were performed by the
enzyme-linked immunosorbent assay (ELISA) and fluorescence
activated cell sorter (FACS) described below, or by measuring the
activity to induce cell death in carcinoma cells.
[0212] By ELISA described in Examples 4 and 5 and the FACS analysis
described in Example 6, a large number of hybridomas producing
human monoclonal antibodies that have human immunoglobulin .gamma.
chain (hIg .gamma.) and human immunoglobulin light chain .kappa.,
and have reactivity specifically to human TRAIL-R1 and/or R2 were
obtained. Furthermore, in any of the following examples including
this example, and tables and figures showing the test results of
the examples, hybridoma clones producing each of the human
anti-human TRAIL-R1 and R2 monoclonal antibodies of the present
invention were denoted using symbols. A clone represented by
symbols with the term "antibody" placed before or after the symbols
means an antibody that is produced by each of the hybridomas, or a
recombinant antibody that is produced by a host cell carrying an
antibody gene (full-length or a variable region) isolated from the
hybridoma. In addition, within a contextually clear range, the name
of a hybridoma clone may express the name of an antibody. The
following hybridoma clones represent single clones: 1-13, 1-18,
1-32, 1-40, 1-43, 2-6, 2-11, 2-12, 2-18, 2-47, 2-52, 3-1, 3-7,
3-10, 3-23, 3-33, 3-42, 3-53, 1-13-6, 1-32-9, 1-40-4, 1-43-43,
2-6-48, 2-11-5, 2-12-10, 2-47-11, 2-52-12, 3-10-19, 3-23-8, 3-33-7,
3-42-3, 3-53-15, 2-18-2, 3-1-7, E-11, E-14, L-30, N-18, X-14,
E-11-13, E-14-4, F-4-2, F-4-8, H-48-2, L-30-10, N-18-12, W-40-5,
X-14-4, X-51-4, X-51-12, A-11, G-3, H-34, I-22, I-35, J-21, J-26,
K-8, K-16, K-57, L-4, P-28, P-36, W-42, X-13, X-60, Z-23, 1-39,
A-4-27, A-4-29, G-3-10, H-34-2, K-57-12, W-42-2, 0304, 0322, KMTR1
and DIM. H-48-2 of these clones was internationally deposited with
International Patent Organism Depositary at the National Institute
of Advanced Industrial Science and Technology (Central 6, 1-1-1,
Higashi, Tsukuba, Ibaraki, Japan) on May 18, 2001. The
international accession number is FERM BP-7599. Furthermore,
E-11-13, F-4-8 and L-30-10 were internationally deposited with the
above deposition center on Aug. 8, 2001. The international
accession number of E-11-13 is FERM BP-7698, that of F-4-8 is FERM
BP-7699, and that of L-30-10 is FERM BP-7700. Furthermore, E-11-13,
F-4-8 and L-30-10 were internationally deposited with the above
deposition center on Oct. 11, 2001. The international accession
number of E-11-13 is FERM BP-7770, that of F-4-8 is FERM BP-7768,
and that of L-30-10 is FERM BP-7769. Furthermore, 0304 and KMTR1
were internationally deposited with the above deposition center on
May 10, 2002. The international accession number of 0304 is FERM
BP-8037, and that of KMTR1 is FERM BP-8038.
Example 4
Detection of Human Anti-TRAIL-R1 Monoclonal Antibody or Human
Anti-TRAIL-R2 Monoclonal Antibody Having Human Immunoglobulin Light
Chain .kappa. (Ig.kappa.)
[0213] Fusion proteins of the extracellular regions of human
TRAIL-R1 and R2 and the Fc region of human IgG1 (hereinafter
referred to as "TRAIL-R1-hFc" and "TRAIL-R2-hFc" (ALEXIS).
Regarding TRAIL-R2-hFc, a region comprising 1 to 183 amino acids
was also used as the extracellular region of TRAIL-R2) were added
at 0.5 .mu.g/ml in phosphate buffered saline (hereinafter referred
to as "PBS"). 50 .mu.l of the thus prepared solution was added to
each well of a 96-well microplate for ELISA (Maxisorp, Nunc) and
incubated for 1 hour at room temperature or at 4.degree. C.
overnight, thereby coating TRAIL-R1-hFc or TRAIL-R2-hFc to the
microplate. Subsequently the supernatant was discarded, a blocking
reagent (SuperBlock (registered trademark) Blocking Buffer, PIERCE)
was added to each well, and then incubation was performed at room
temperature for 30 minutes, thereby blocking the part where
TRAIL-R1-hFc or TRAIL-R2-hFc did not bind. Thus, a microplate
having each well coated with TRAIL-R1 -hFc or TRAIL-R2-hFc was
prepared.
[0214] The culture supernatant of each hybridoma (50 .mu.l) was
added to each well, reaction was performed at room temperature for
1 hour, and then each well was washed twice in 0.1%
Tween20-containing PBS (PBS-T). Subsequently, horseradish
peroxidase-labeled goat anti-human Ig.kappa. antibodies (50
.mu.l/well, Biosource International) were diluted 2000 times in
PBS-T containing 10% Block Ace (Dainippon Pharmaceutical Co.,
Ltd.). 50 .mu.l of the thus prepared solution was added to each
well, and incubation was then performed at room temperature for 30
minutes. The microplate was washed three times with PBS-T, and then
100 .mu.l of a TMB chromogenic substrate solution (DAKO) was added
to each well, followed by incubation at room temperature for 20
minutes. 0.5M sulfuric acid was added (100 .mu.l/well) to each well
to stop reaction. Absorbance at a wavelength of 450 nm (reference
wavelength of 570 nm) was measured with a microplate reader
(MTP-300, Corona Electric). Moreover, antibodies produced by the
hybridomas 0304, 0322, KMTR1 and DIM were subjected to the above
experiment using the purified antibodies obtained by the method
described in Example 10.
[0215] Table 1 and Table 2 show the characteristics of the part of
antibodies among the thus obtained anti-human TRAIL-R1 and R2
antibodies. Table 1 shows the subclass and cross reactivity of the
obtained human anti-TRAIL-R1 monoclonal antibodies. Table 2 shows
the subclass and cross reactivity of the obtained human
anti-TRAIL-R2 monoclonal antibodies.
2TABLE 1 Human anti-TRAIL-R1 Cross reactivity antibody Subclass
TRAIL-R1 TRAIL-R2 1-13 IgG4 + - 1-18 IgG4 + - 1-32 IgG1 + - 1-40
IgG1 + - 1-43 IgG1 + - 2-6 IgG1 + - 2-11 IgG1 + - 2-12 IgG1 + -
2-18 IgM + - 2-47 IgG4 + - 2-52 IgG1 + - 3-1 IgM + - 3-7 IgM + -
3-10 IgG4 + - 3-23 IgG4 + - 3-33 IgG4 + - 3-42 IgG2 + - 3-53 IgG1 +
- +: with reactivity -: no reactivity
[0216]
3TABLE 2 Human anti-TRAIL-R2 Cross reactivity antibody Subclass
TRAIL-R1 TRAIL-R2 A-4-27 IgM - + A-4-29 IgM + + A-11 IgM - + E-11
IgG1 - + E-14 IgG1 - + F-4-2 IgG4 - + F-4-8 IgG1 - + G-3 IgM - +
H-34 IgM - + H-48-2 IgG1 - + I-22 IgM - + I-35 IgM - + J-21 IgM - +
J-26 IgM - + K-8 IgM - + K-16 IgM - + K-57 IgM - + L-4 IgM - + L-30
IgG1 - + N-18 IgG4 - + P-28 IgM - + P-36 IgM - + W-40-5 IgG1 - +
W-42 IgM - + X-13 IgM - + X-14 IgG4 - + X-51-4 IgG1 - + X-51-12
IgG4 - + X-60 IgM - + Z-23 IgM - + 1-39 IgM - + 0304 IgG4 - + 0322
IgG4 - + KMTR1 IgG1 + + D1M IgG1 + + +: with reactivity -: no
reactivity
Example 5
Identification of the Subclass of Each Monoclonal Antibody
[0217] A microplate having each well coated with TRAIL-R1-hFc or
TRAIL-R2-hFc was prepared by a method similar to that of Example 4,
and then each well was washed twice with PBS-T. The culture
supernatant (50 .mu.l) of each of the hybridomas obtained in
Example 4 was added to each well of the microplate coated with
TRAIL-R1-hFc or TRAIL-R2-hFc to perform reaction for 1 hour, and
then each well was washed twice with PBS-T. Subsequently, sheep
anti-human IgG1 antibodies, sheep anti-human IgG2 antibodies, or
sheep anti-human IgG3 antibodies or sheep anti-human IgG4
antibodies, which had been respectively labeled with horseradish
peroxidase and diluted 2000 times, were added (50 .mu.l/well, The
Binding Site) to each well, followed by incubation at room
temperature for 1 hour. After washing 3 times with PBS-T, a
substrate buffer (TMB, 100 .mu.l/well, DAKO) was added to each
well, and then incubation was performed at room temperature for 20
minutes. Next, 0.5M sulfuric acid (100 .mu.l/well) was added to
stop the reaction. Absorbance at a wave length of 450 nm (with a
reference wavelength of 570 nm) was measured using a microplate
reader (MTP-300, Corona Electric). In addition, antibodies produced
by the hybridomas 0304, 0322, KMTR1 and DIM were subjected to the
above experiment using the purified antibodies obtained by the
method of Example 10. The above Table 1 and Table 2 show the
results.
Example 6
Test of the Reactivity of Each Monoclonal Antibody to TRAIL-R1 and
R2 Expressing Cells
[0218] The reactivity of each of the monoclonal antibodies obtained
in Example 4 to the TRAIL-R1delta-expressing L929 cells and
TRAIL-R2delta-expressing L929 cells prepared in Example 1 was
examined by FACS analysis. L929 cells, TRAIL-R1delta-expressing
L929 cells and TRAIL-R2delta-expressing L929 cells were suspended
at a concentration of 2.times.10.sup.6/ml in a staining buffer (SB)
of PBS containing 1% rabbit serum, 0.1% NaN.sub.3 and 1% FCS. The
cell suspension (100 .mu.l/well) was added into a 96-well
round-bottomed plate (Beckton Dickinson). After centrifugation
(2000 rpm, 4.degree. C., 2 minutes), the supernatant was removed
and then the culture supernatant (50 .mu.l) of the hybridoma
cultured in Example 3 was added. The mixture was agitated, allowed
to stand on ice for 30 minutes, and then subjected to
centrifugation (2000 rpm, 4.degree. C. for 2 minutes) to remove the
supernatant. After the pellet was washed twice with SB (100
.mu.l/well), 30 .mu.l of 0.0125 mg/ml RPE fluorescence-labeled
rabbit anti-human Ig.kappa. F(ab').sub.2 antibodies (DAKO) was
added, and then incubation was performed on ice for 30 minutes.
After washed twice with SB, the cells were suspended in 300 .mu.l
of SB, and then fluorescence intensity of each cell was measured by
FACS (FACScan, Beckton Dickinson). As a result, all the antibodies
were observed to have strong binding activity only to the
TRAIL-R1delta-expressing L929 cells or the TRAIL-R2delta-expressing
L929 cells, and no binding activity to L929 cells was observed.
Thus, it was shown that they were antibodies binding specifically
to TRAIL-R1 and TRAIL-R2.
Example 7
Cell-death-inducing Activity on Carcinoma Cells
[0219] Using the culture supernatant of the hybridoma producing the
human anti-TRAIL-R1 monoclonal antibodies or the human
anti-TRAIL-R2 monoclonal antibodies obtained from Example 3 or 4 to
6, cell-death-inducing activity on Colo205 (ATCC No. CCL-222)
cells, which were colon carcinoma cells, was measured. Colo205
cells cultured in RPMI media containing 10% FCS were prepared at a
concentration of 2.5.times.10.sup.4/ml. 100 .mu.l of the suspension
was added to each well of a 96-well flat bottomed plate (Beckton
Dickinson). After culturing at 37.degree. C. under 5.0% carbon
dioxide gas for 24 hours, the hybridoma culture supernatant was
added at 50 .mu.l/well. Furthermore, when the human anti-TRAIL-R1
monoclonal antibody or the human anti-TRAIL-R2 monoclonal antibody
was IgG, goat anti-human IgG (.gamma.)-specific polyclonal
antibodies (Sigma) were added (10 .mu.l/well) to each well at a
final concentration of 5 .mu.g/ml. For a part of the obtained
hybridomas, wells not supplemented with goat anti-human IgG
(.gamma.)-specific polyclonal antibodies were prepared. As a
positive control, human recombinant TRAIL protein (DAKO) was
employed with a final concentration of 100 ng/ml. As a negative
control, human IgG (Biogenesis) was employed. After 48 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas, an MTS
reagent (Cell Titer 96 AQ.sub.UEOUS Non-Radioactive Cell
Proliferation Assay: Promega) was prepared according to the method
described in the instructions, and then 20 .mu.l of the reagent was
added to each well. After another 2 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, absorbance at a
wavelength of 490 nm (reference wavelength of 630 nm) was measured
using a microplate reader (1420 ARVO multi-label counter: WALLAC).
Using the reducibility of mitochondria as an indicator, the
survival rate of the cells was calculated. The survival rate of the
cells in each well was calculated by the following formula:
Survival rate (%)=100.times.(a-b)/(c-b) (wherein "a" represents the
measured value of a well tested, "b" represents the measured value
of a cell-free well, and "c" represents the measured value of a
negative control well). FIGS. 1 to 3 and Tables 3 and 4 show the
results. Table 3 shows the cell-death-inducing activity (in the
culture supernatant of the hybridomas producing the human
anti-TRAIL-R1 monoclonal antibodies) on Colo205 and normal human
hepatocytes. Table 4 shows the cell-death-inducing activity (in the
culture supernatant of the hybridomas producing the human
anti-TRAIL-R2 monoclonal antibodies) on Colo205 and human normal
heptocytes.
4TABLE 3 Normal human Human anti-TRAIL-R1 hepatocyte Colo205 cell
antibody Subclass survival rate survival rate 1-13-6 IgG4 - -
1-32-9 IgG1 - - 1-40-4 IgG1 - - 1-43-43 IgG1 - - 2-6-48 IgG1 - -
2-11-5 IgG1 ++ ++ 2-12-10 IgG1 - - 2-47-11 IgG4 + + 2-52-12 IgG1 ++
++ 3-10-19 IgG4 - - 3-23-8 IgG4 - - 3-33-7 IgG4 - - 3-42-3 IgG2 - -
3-53-15 IgG1 - - 2-18-2 IgM ++ ++ 3-1-7 IgM - + sTRAIL 1 .mu.g/ml
-- - - ++: Survival rate of 80% or more +: Survival rate of 21% to
79% -: Survival rate of 20% or less
[0220]
5TABLE 4 Normal human Human anti-TRAIL-R2 hepatocyte Colo205 cell
antibody Subclass survival rate survival rate E-11-13 IgG1 ++ -
E-14-4 IgG1 + + F-4-2 IgG4 + - F-4-8 IgG1 - - H-48-2 IgG1 ++ -
L-30-10 IgG1 ++ - N-18-12 IgG4 ++ - W-40-5 IgG1 ++ + X-14-4 IgG4 ++
+ W-51-4 IgG1 - - X-51-12 IgG4 ++ - A-4-29 IgM - - G-3-10 IgM ++ -
H-34-2 IgM - - K-57-12 IgM + - W-42-2 IgM - - sTRAIL 1 .mu.g/ml --
- - ++: Survival rate of 80% or more +: Survival rate of 21% to 79%
-: Survival rate of 20% or less
[0221] As a result, it was revealed that the human anti-TRAIL-R1
and R2 monoclonal antibodies clearly had activity to induce cell
death in Colo205 cells, compared with the negative control.
Moreover, it was shown that a part of the human anti-TRAIL-R2
monoclonal antibodies, which is IgG, had activity to induce cell
death even in the absence of goat anti-human IgG(.gamma.)-specific
polyclonal antibodies (in a state without cross-linking with the
human anti-TRAIL-R2 monoclonal antibodies).
Example 8
Cell-death-inducing Activity on Normal Cells
[0222] Cell-death-inducing activity on HUVEC (Biowhittaker), which
is a normal human umbilical vein endothelial cell, was measured
using the culture supernatant of the hybridomas producing the human
anti-TRAIL-R2 monoclonal antibodies obtained in Examples 4 to 6.
HUVEC cells cultured in an EGM-2 medium (Biowhittaker) were
prepared at a concentration of 5.times.10.sup.4/ml. 100 .mu.l of
the suspension was added to each well of a 96-well flat-bottomed
plate (Beckton Dickinson). The cells were cultured at 37.degree. C.
under 5.0% carbon dioxide gas for 24 hours, and then the culture
supernatant of the hybridoma was added at 50 .mu.l/well. Further,
when the human anti-TRAIL-R1 monoclonal antibody or the human
anti-TRAIL-R2 monoclonal antibody was IgG, 10 .mu.l of goat
anti-human IgG(.gamma.)-specific polyclonal antibodies (Sigma) were
added at a final concentration of 5 .mu.g/ml to each well. Human
IgG (Biogenesis) was used as a negative control. After 48 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas, an MTS
reagent (Cell Titer 96 AQ.sub.UEOUS Non-Radioactive Cell
Proliferation Assay: Promega) was prepared according to the method
described in the instructions, and then 20 .mu.l of the reagent was
added to each well. After another 2 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, absorbance at a
wavelength of 490 nm (with a reference wavelength of 630 nm) was
measured using a microplate reader (1420 ARVO multi-label counter:
WALLAC). Using the reducibility of mitochondria as an indicator,
the survival rate of the cells was calculated. The survival rate of
the cells of each well was calculated by a formula similar to that
of Example 7.
[0223] FIG. 4 shows the result. The human anti-TRAIL-R2 monoclonal
antibody and the negative control showed almost the same result,
revealing that the human anti-TRAIL-R2 monoclonal antibody does not
show cytotoxicity against HUVEC cells.
Example 9
Cell-death-inducing Activity on Normal Human Hepatocytes
[0224] Cell-death-inducing activity on normal human hepatocytes
(hereinafter referred to as "HH cells") (Tissue Transformation
Technologies) was measured using the culture supernatant of the
hybridomas producing the human anti-TRAIL-R1 and R2 monoclonal
antibodies obtained in Examples 4 to 6. First, frozen HH cells were
thawed at 37.degree. C., and then prepared at a concentration of
7.5.times.10.sup.5/ml using a CM5300 medium (CEDRA). 100 .mu.l of
the suspension was added to each well of a 96-well flat-bottomed
plate coated with collagen type I (Beckton Dickinson). After 4.5
hours of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
medium exchange was performed. After 24 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, medium exchange was
performed again. Subsequently, the culture supernatant of the
hybridoma was added at 50 .mu.l/well, and then 10 .mu.l of goat
anti-human IgG(.gamma.)-specific polyclonal antibodies (Sigma) were
added to each well at a final concentration of 5 .mu.g/ml. Human
IgG (Biogenesis) was used as a negative control. After 24 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas,
morphological changes in HH cells were observed under a microscope.
The result of the human anti-TRAIL-R2 monoclonal antibody and that
of negative control were almost the same, revealing that the human
anti-TRAIL-R2 monoclonal antibody does not show cytotoxicity also
against HH cells.
Example 10
Preparation of Each Antibody
[0225] The human anti-TRAIL-R1 and R2 monoclonal antibodies from
the culture supernatant of the hybridomas obtained from Examples 4,
6 and 7 were purified by the following method. The culture
supernatant containing the human anti-TRAIL-R1 and R2 monoclonal
antibodies was subjected to affinity purification using rmp Protein
A (Amersham Pharmacia Biotech), a 0.8.times.40 cm column (Bio-Rad),
PBS as an adsorption buffer and a 0.02 M glycine buffer (pH 3) as
an elution buffer. The eluted fraction was adjusted to have a pH of
around 7.2 by adding 1 M Tris (pH 9.0). The thus prepared antibody
solution was substituted with PBS using a dialysis membrane (10000
cut, Spectrum Laboratories), and then filtered using a MILLEX-GV
membrane filter (Millipore) with a pore size of 0.22 .mu.m for
sterilization, thereby obtaining purified human anti-TRAIL-R1 and
R2 monoclonal antibodies. Absorbance at 280 nm was measured, and
then the concentration of the purified antibodies was calculated
using 1.4 OD=1 mg/ml.
[0226] The culture supernatant containing the human anti-TRAIL-R1
and R2 monoclonal antibodies was prepared by the following method.
First, human anti-TRAIL-R1 and R2 monoclonal antibodies-producing
hybridomas were adapted in an eRDF medium (Kyokutoseiyaku)
containing 10 ng/ml Recombinant Human IL-6 (R&D Systems) and
10% Low IgG fetal bovine serum (HyClone). The adapted hybridomas
were cryopreserved. Next, for the purpose of antibody purification,
a part of the hybridomas was adapted in an eRDF medium
(Kyokutoseiyaku) containing bovine insulin (5 .mu.g/ml, Gibco BRL),
human transferrin (5 .mu.g/ml, Gibco BRL), ethanolamine (0.01 mM,
Sigma), sodium selenite (2.5.times.10.sup.5 mM, Sigma), 10 ng/ml
recombinant human IL-6 (R&D Systems) and 1% Low IgG fetal
bovine serum (HyClone). The hybridoma cells were cultured in
flasks, and when the viable cell ratio of the hybridoma reached
90%, the culture supernatant was collected. The collected
supernatant was applied to a 10 .mu.m filter and a 0.2 .mu.m filter
(German Science), thereby removing miscellaneous waste materials
such as hybridomas.
Example 11
Cell-death-inducing Activity of Purified Human Anti-TRAIL-R2
Monoclonal Antibody on Carcinoma Cells and Normal Human
Hepatocytes
[0227] The cell-death-inducing activity on the colon carcinoma cell
Colo205 (ATCC No. CCL-222) was measured using the purified human
anti-TRAIL-R2 monoclonal antibodies obtained in Example 10. Colo205
cells cultured in RPMI media containing 10% FCS were prepared at a
concentration of 2.5.times.10.sup.4/ml, and then the 100 .mu.l of
the suspension was added to each well of a 96-well flat-bottomed
plate (Beckton Dickinson). After 24 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, the purified
antibodies were added (10 .mu.l/well) at final concentrations of
10, 100 and 1000 ng/ml. Further, 10 .mu.l of goat anti-human IgG
(.gamma.)-specific polyclonal antibodies (Sigma) were added to each
well at a final concentration of 10 .mu.g/ml. For a part of the
obtained hybridomas, wells not supplemented with goat anti-human
IgG (.gamma.)-specific polyclonal antibodies were prepared. As a
positive control, human recombinant TRAIL proteins (R&D
SYSTEMS) with final concentrations of 0.1, 1 and 10 ng/ml were
used. A human anti-HSA antibody was used as a negative control.
Culturing was performed at 37.degree. C. under 5.0% carbon dioxide
gas for 48 hours, so as to cause the antibodies to react with the
receptors on the cell surfaces. The volume per reaction system was
120 .mu.l. In addition, for 0304 and KMTR1, an experiment wherein
no goat anti-human IgG (.gamma.)-specific monoclonal antibodies
were added as the cross-linker was conducted (described as "alone"
in Table 5). The volume per reaction system in this case was 110
.mu.l. After culturing, an MTS reagent (Cell Titer 96 AQ.sub.UEOUS
Non-Radioactive Cell Proliferation Assay: Promega) was prepared
according to the method described in the instructions. 20 .mu.l of
the reagent was added to each well. After 2 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, absorbance at a
wavelength of 490 nm (with a reference wavelength of 630 nm) was
measured using a microplate reader (1420 ARVO multi-label counter:
WALLAC). Using the reducibility of the mitochondria as an
indicator, the survival rate of the cells was calculated. The
survival rate of the cells in each well was calculated using a
formula similar to that of Example 7.
[0228] Next, the cell-death-inducing activity on HH cells (Tissue
Transformation Technologies, and In Vitro Technologies) was
measured using the human anti-TRAIL-R2 monoclonal antibodies
obtained in Example 10. First the frozen HH cells were thawed at
37.degree. C., and then prepared at a concentration of
7.5.times.10.sup.5/ml using a CM5300 medium (CEDRA). 100 .mu.l of
the suspension was added to each well of a 96-well flat-bottomed
plate coated with collagen type I (Beckton Dickinson). After 4.5
hours of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
medium exchange was performed. After another 24 hours of culturing
at 37.degree. C. under 5.0% carbon dioxide gas, the medium was
exchanged with a serum-free medium [DMEM medium (Sigma) containing
insulin (20 .mu.g/ml, Sigma), glucagon (7 ng/ml, Sigma),
hydrocortisone (7.5 .mu.g/ml, Sigma) and human EGF (20 ng/ml,
Beckton Dickinson)] or a CM5300 medium. Subsequently, the purified
antibodies were added (10 .mu.l/well) at final concentrations of
0.1, 1 and 10 .mu.g/ml, and then 10 .mu.l of goat-anti-human
IgG(.gamma.)-specific polyclonal antibodies (Sigma) were added to
each well at final concentrations of 10 and 100 .mu.g/ml. For a
part of the obtained hybridomas, wells not supplemented with goat
anti-human IgG(.gamma.)-specific polyclonal antibodies were
prepared. As a negative control, human anti-HSA antibodies were
used. Culturing was performed at 37.degree. C. under 5.0% carbon
dioxide gas for 24 hours, the antibodies and the receptors on the
cell surfaces were allowed to react. The volume per reaction system
was 120 .mu.l. In addition, for 0304 and KMTR1, an experiment
wherein no goat anti-human IgG(.gamma.)-specific monoclonal
antibodies were added as the cross-linker was conducted (described
as "alone" in Table 5). The volume per reaction system in this case
was 110 .mu.l. After culturing, HH cells were washed twice with
PBS, 100 .mu.l of PBS was added to each well, and then Triton X-100
was added (10 .mu.l/well) at a final concentration of 0.8%. The
cells were allowed to stand at 37.degree. C. for 1 hour, so that
living HH cells were lysed. The lysate was transferred (50
.mu.l/well) to a different 96-well flat-bottomed plate, and then
subjected to LDH assay. A reagent for LDH assay (CytoTox 96
Non-Radioactive Cytotoxicity Assay: Promega) was prepared according
to the method described in the instructions, and then 50 .mu.l of
the reagent was added to each well. The plate was protected from
light, and then it was allowed to stand at room temperature for 30
minutes. A reaction stop solution (1M acetic acid: Promega) was
added at 50 .mu.l/well. Absorbance at a wavelength of 492 nm was
measured using a microplate reader (1420 ARVO multi-label counter:
WALLAC). The survival rate of the cells was calculated using the
enzymatic activity of LDH as an indicator. The survival rate of the
cells of each well was calculated by a formula similar to that of
Example 7.
[0229] Furthermore, LD50 values were calculated by the following
method using the calculated survival rate. In the graph, the
calculated survival rates at each antibody concentration are
plotted on the longitudinal axis and the concentrations of the
antibodies added to the cells are plotted on the horizontal axis.
Plotted dots adjacent to each other are connected to make a curve.
A formula expressing this curve was found by a regression
calculation. The antibody concentrations corresponding to the
survival rate of 50% were calculated using the formula, thereby
obtaining LD50 values.
[0230] FIG. 5a to l and Table 5 show the results. In FIG. 5, a
solid line with solid circles (--.circle-solid.--) expresses normal
human hepatocyte, and a dotted line with solid diamond-shaped
symbols (--.diamond-solid.--) expresses Colo205 cells. Furthermore,
FIG. 5k and l show the results of experiments wherein no goat
anti-human IgG(.gamma.)-specific polyclonal antibodies were added.
Table 5 shows the cell-death-inducing activity (LD50 value) of the
purified human anti-TRAIL-R2 monoclonal antibody on colon carcinoma
cells Colo205, and normal human hepatocytes. 2.5.times.10.sup.3
colon carcinoma cells Colo205, were seeded in 100 .mu.l of a medium
per well of a 96-well flat-bottomed plate, and the purified human
anti-TRAIL-R2 monoclonal antibodies were added to the cells on the
next day. When the time for the reaction between the cells and the
antibody reached 48hours, the LD50 value was obtained.
7.5.times.10.sup.4 normal cells (human hepatocytes) were seeded in
100 .mu.l of a medium per well of a 96-well flat-bottomed plate,
and the purified human anti-TRAIL-R2 monoclonal antibodies were
added to normal cells (human hepatocytes) on the next day. When the
time for the reaction between the cells and antibody reached to 24
hours, the LD50 value was obtained. Compared with the negative
control, the purified human anti-TRAIL-R2 monoclonal antibody was
shown to clearly have activity to induce cell death in Colo205
cells. Furthermore, compared with the human recombinant TRAIL and
purified antibody H-48-2, the human hepatocyte toxicity of the
purified human anti-TRAIL-R2 monoclonal antibodies E-11-13, L-30-10
and KMTR1 were shown to be low.
[0231] Moreover, KMTR1 was shown by the results of Example 4 to
bind to both receptors, TRAIL-R1 and TRAIL-R2. It can be expected
that this antibody can transduce cell-death-inducing signals via
either TRAIL-R1 or TRAIL-R2 receptor.
[0232] Since the hepatocytes showed a survival rate of 50% or more
even when 10 .mu.g/ml L-30-10 was added, the LD50 of L-30-10 was 10
.mu.g/ml or more. The LD50 was 24 .mu.g/ml when regression
calculation was performed based on a graph on which the antibody
concentrations and the survival rates had been plotted. Since the
hepatocytes showed a survival rate of 50% or less when 0.1 .mu.g/ml
F-4-8 was added, the LD50 of F-4-8 was 0.1 .mu.g/ml or less. The
LD50 was 0.002 .mu.g/ml based on a regression calculation performed
similarly to that for L-30-10. The LD50 values of KMTR1 and DIM
were both confirmed to be 10 .mu.g/ml or more. Further in a case
where no goat anti-human IgG(.gamma.)-specific polyclonal
antibodies were added (hereinafter referred to as "in the "alone"
case"), the survival rate of hepatocytes was never below 50%, even
when KMTR1 with an antibody volume of 100 .mu.g/ml was added. Thus,
it was confirmed that the LD50 in the "alone" case was 100 .mu.g/ml
or more.
[0233] Next, the ratio of the LD50 value for normal hepatocytes to
that for Colo 205 (showing how many times the LD50 value for normal
human hepatocytes is greater than that for Colo205 cells) was
measured (N/C ratio). The results were N/C=25.45 (10 times or more
greater) in the case of purified antibody E-11-3, N/C=67 or more
(10 times or more greater) in the case of DIM, N/C=50 (10 times or
more greater) in the case of 0304 alone, N/C=240 (100 times or more
greater) in the case of L-30-10, and N/C=1000 times or more greater
in the case of KMTR1 alone. Thus, all the antibodies were shown to
be excellent in terms of efficacy and safety (Table 5).
6TABLE 5 Purified human Normal human anti-TRAIL-R2 hepatocyte
Colo205 antibody LD50 (.mu.g/ml) LD50 (.mu.g/ml) N/C ratio E-11-13
2.8 0.11 25.45 F-4-8 0.002 0.02 0.1 H-48-2 0.12 0.15 0.8 L-30-10 24
0.1 240 W-40-5 7.47 0.7 10.7 0304 0.002 0.02 0.1 0322 0.06 0.04 1.5
KMTR1 >10 0.04 >250 D1M >10 0.15 >67 0304 (alone) 1
0.02 50 KMTR1 (alone) >100 0.1 >1000 Human recombinant 0.25
ng/ml 2 ng/ml 0.125 TRAIL
[0234] By a similar method, the cell-death-inducing activity of the
purified human anti-TRAIL-R2 monoclonal antibodies was examined for
U251 cells (derived from glioma, Riken Genebank No. RCB0461) and
Jurkat cells (derived from T cell lymphomas, Dainippon
Pharmaceutical Co., Ltd.). In an experiment for U251 cells,
1.0.times.10.sup.4 cells were seeded in 100 .mu.l of a medium per
well of a 96-well flat-bottomed plate and then cultured at
37.degree. C. in the presence of 5% CO.sub.2. The antibodies were
added on the next day. After culturing under the above environment
for 48 hours, the survival rate of the cells was measured. In an
experiment for Jurkat cells, 4.0.times.10.sup.4 cells were seeded
in 100 .mu.l of a medium per well of a 96-well flat-bottomed plate,
and then the antibodies were added. After 48 hours of culturing at
37.degree. C. in the presence of 5% CO.sub.2, the survival rate of
the cells was measured. The LD50 value (unit: .mu.g/ml) of each
antibody is as shown below.
[0235] LD50 of E-11-13 for U251 cells: 0.3, and for Jurkat cells:
0.1.
[0236] LD50 of L-30-10 for U251 cells: 0.17, and for Jurkat cells:
0.13.
[0237] LD50 of H-48-2 for U251 cells: 0.24, and for Jurkat cells:
0.07.
[0238] LD50 of F-4-8 for U251 cells: 0.03, and for Jurkat cells:
0.004.
[0239] LD50 of W-40-5 for U251 cells: 1.0, and for Jurkat cells:
0.48.
[0240] In addition for U251 cells, assay was performed with a
system wherein a cisplatin solution (NIPPON KAYAKU) with a final
concentration of 4 .mu.g/ml was added simultaneously with the
antibody.
Example 12
Effect of Purified Human Anti-TRAIL-R2 Monoclonal Antibodies on
Tumor-bearing mice
[0241] The effect of the human anti-TRAIL-R2 monoclonal antibody
obtained in Example 10 was examined using a tumor-bearing mouse
model according to the following method.
[0242] Colo205, colon carcinoma cells, were subcutaneously
transplanted in the dorsal areas at 5.times.10.sup.6/mouse to 4- to
6-week-old Balb/c nude mice (purchased from CLEA Japan). 1 week to
10 days after transplantation, the sizes of tumors that had adhered
were measured. 5 or 7 tumor-bearing mice having average tumor sizes
of approximately 100 mm.sup.3 or 300 mm.sup.3 were grouped into a
single group. Into the peritoneal cavities of the tumor-bearing
mice, the purified antibodies were administered at 1, 4, 20, 25 and
100 .mu.g/mouse (dissolved in 200 .mu.l of PBS), and then the tumor
size was measured. The same volume of human anti-HSA antibodies was
used as a negative control of the antibody.
[0243] FIGS. 6 to 10 show the results of the above experiments. In
the groups where purified human anti-TRAIL-R2 monoclonal antibodies
E-11-13, F-4-8, H-48-2, L-30-10 and W-40-5 had been administered at
1 .mu.g/mouse, a regression effect was observed in the group to
which H-48-2 had been administered. The anti-tumor effects were
lower in descending order of E-11-13, L-30-10, F-4-8 and W-40-5
(FIG. 6). In FIG. 6, when the antibody was administered 3 times on
alternate days, growth suppression and a regression effect were
observed at least for 13 days when calculated from the initial
administration (H-48-2 clone).
[0244] In the groups to which E-11-13 had been administered at 4,
20 and 100 .mu.g/mouse, anti-tumor effects were confirmed in all
the mice. With a dose of 20 .mu.g/mouse, the highest tumor
regression effect was observed (FIG. 7). In FIG. 7, when the
antibody was administered 4 times on alternate days, growth
suppression and a regression effect were observed at least for 11
days when calculated from the initial administration. Changes with
time in tumor volume of the group to which the antibodies were
administered at 20 .mu.g/mouse 4 times on alternate days
(administered on days 7, 9, 11 and 13 after transplantation) were
as follows.
[0245] On day 2 after the initial administration (corresponding to
day 9 in FIG. 7), the average tumor volume was 109.5 mm.sup.3;
[0246] On day 4 after the initial administration (corresponding to
day 11 in FIG. 7), the average tumor volume was 85.1 mm.sup.3:
[0247] On day 6 after the initial administration (corresponding to
day 13 in FIG. 7), the average tumor volume was 64.3 mm.sup.3:
[0248] On day 8 after the initial administration (corresponding to
day 15 in FIG. 7), the average tumor volume was 61.8 mm.sup.3;
and
[0249] On day 11 after the initial administration (corresponding to
day 18 in FIG. 7), the average tumor volume was 78.9 mm.sup.3.
[0250] The tumor volume on day 4 after the start of administration
was approximately 85.1 mm.sup.3, and a 14% or more tumor reduction
was observed. This reduction was maintained on day 11 after the
administration, showing that the antibody of the present invention
possesses a high anti-tumor effect.
[0251] E-11-3 was administered at 20 .mu.g/mouse to a group of 7
tumor-bearing mice where the tumors were approximately 300 mm.sup.3
on average. As a result, significant tumor regression was observed
(FIG. 8). In FIG. 8, when the antibody was administered 3 times on
alternate days, growth suppression and a regression effect were
observed at least for 18 days when calculated from the initial
administration. Changes with time in tumor volume of the group to
which the antibodies were administered at 20 .mu.g/mouse 3 times on
alternate days (administered on days 9, 11 and 13 after
transplantation) are as follows.
[0252] On day 2 after the initial administration (corresponding to
day 11 in FIG. 8), the average tumor volume was 246.9 mm.sup.3;
[0253] On day 4 after the initial administration (corresponding to
day 13 in FIG. 8), the average tumor volume was 181.8 mm.sup.3;
[0254] On day 5 after the initial administration (corresponding to
day 14 in FIG. 8), the average tumor volume was 146.2 mm.sup.3;
[0255] On day 6 after the initial administration (corresponding to
day 15 in FIG. 8), the average tumor volume was 110.8 mm.sup.3;
[0256] On day 7 after the initial administration (corresponding to
day 16 in FIG. 8), the average tumor volume was 82.7 mm.sup.3;
[0257] On day 9 after the initial administration (corresponding to
day 18 in FIG. 8), the average tumor volume was 57.5 mm.sup.3;
[0258] On day 11 after the initial administration (corresponding to
day 20 in FIG. 8), the average tumor volume was 81.3 mm.sup.3;
[0259] On day 13 after the initial administration (corresponding to
day 22 in FIG. 8), the average tumor volume was 108.1 mm.sup.3;
[0260] On day 15 after the initial administration (corresponding to
day 24 in FIG. 8), the average tumor volume was 127.8 mm.sup.3;
and
[0261] On day 18 after the initial administration (corresponding to
day 27 in FIG. 8), the average tumor volume was 163.3 mm.sup.3.
[0262] The tumor volume on day 4 after the start of administration
was approximately 181.8 mm.sup.3, and a 39% or more tumor reduction
was observed. This reduction was maintained even on day 18 after
the administration, showing that the antibody of the present
invention possesses a high anti-tumor effect.
[0263] The activity of 0304 antibody was evaluated as follows.
Colo205, colon carcinoma cells, were subcutaneously transplanted in
the dorsal areas at 5.times.10.sup.6/mouse to 6-week-old Balb/c
nude mice (purchased from CLEA Japan). 8 days after
transplantation, the sizes of tumors that had adhered were
measured. 5 tumor-bearing mice having an average tumor size of
approximately 100 mm.sup.3 were grouped into a single group. Into
the peritoneal cavities of the tumor-bearing mice, the purified
antibodies were administered at 20 .mu.g/ mouse (dissolved in 200
.mu.l of PBS), and then the tumor size was measured. Anti-tumor
effects were confirmed in all the mice of the group, to which 0304
had been administered (at 20 .mu.g/mouse) 3 times on alternate days
(administered on days 8, 10 and 12 after transplantation) (FIG. 9).
Changes with time in tumor volume are as follows.
[0264] On day 2 after the initial administration (corresponding to
day 10 in FIG. 9), the average tumor volume was 142.092
mm.sup.3;
[0265] On day 4 after the initial administration (corresponding to
day 12 in FIG. 9), the average tumor volume was 34.138
mm.sup.3;
[0266] On day 7 after the initial administration (corresponding to
day 15 in FIG. 9), the average tumor volume was 18.641 mm.sup.3;
and
[0267] On day 11 after the initial administration (corresponding to
day 19 in FIG. 9), the average tumor volume was 9.339 mm.sup.3;
[0268] The tumor volume on day 4 after the start of administration
was approximately 34.138 mm.sup.3, and a 65% or more tumor
reduction was observed. This reduction was maintained even on day
11 after the administration, showing that the antibody of the
present invention possesses an extremely high anti-tumor
effect.
[0269] Next, Colo205, colon carcinoma cells, were subcutaneously
transplanted in the dorsal areas at 5.times.10.sup.6/mouse to
12-week-old Balb/c nude mice (purchased from CLEA Japan). 10 days
after transplantation, the sizes of tumors that had adhered were
measured. 5 tumor-bearing mice having an average tumor size of
approximately 100 mm.sup.3 were grouped into a single group. Into
the peritoneal cavities of the tumor-bearing mice, the purified
antibodies were administered at 25 .mu.g/ mouse (dissolved in 200
.mu.l of PBS), and then the tumor size was measured. As a negative
control of the antibody, the same volume of human anti-HSA
antibodies was used. Anti-tumor effects were confirmed in all the
mice of the group, to which 0304 had been administered at 25
.mu.g/mouse 3 times (administered on days 10, 13 and 15 after
transplantation) (FIG. 10). Changes with time in tumor volume are
as follows.
[0270] On day 3 after the initial administration (corresponding to
day 13 in FIG. 10), the average tumor volume was 54.626
mm.sup.3;
[0271] On day 5 after the initial administration (corresponding to
day 15 in FIG. 10), the average tumor volume was 32.357
mm.sup.3;
[0272] On day 8 after the initial administration (corresponding to
day 18 in FIG. 10), the average tumor volume was 15.895
mm.sup.3;
[0273] On day 12 after the initial administration (corresponding to
day 22 in FIG. 10), the average tumor volume was 14.377
mm.sup.3;
[0274] On day 15 after the initial administration (corresponding to
day 25 in FIG. 10), the average tumor volume was 26.654
mm.sup.3;
[0275] On day 19 after the initial administration (corresponding to
day 29 in FIG. 10), the average tumor volume was 27.565
mm.sup.3;
[0276] On day 25 after the initial administration (corresponding to
day 35 in FIG. 10), the average tumor volume was 30.802
mm.sup.3;
[0277] On day 29 after the initial administration (corresponding to
day 39 in FIG. 10), the average tumor volume was 27.092 mm.sup.3;
and
[0278] On day 32 after the initial administration (corresponding to
day 42 in FIG. 10), the average tumor volume was 32.921
mm.sup.3;
[0279] On day 12 after the initial administration (corresponding to
day 22 in FIG. 10), tumor disappearance was confirmed in 3 out of 5
mice.
[0280] The average tumor volume on day 3 after the initial
administration was 54.626 mm.sup.3, and a 45% or more tumor
reduction was observed. Moreover, the average tumor volume on day 5
after the administration was 32.357 mm , and a 65% or more tumor
reduction was observed. This reduction was maintained on day 32
after the administration. Specifically, the 65% or more reduction
was maintained at least for 27 days. Thus, it was shown that the
antibody of the present invention possesses an extremely high
anti-tumor effect.
[0281] Furthermore, in FIG. 10, growth suppression and a regression
effect were observed for at least 32 days when calculated from the
initial administration.
[0282] In addition, in FIGS. 9 and 10, "Vehicle" represents PBS
(200 .mu.l) that was used as a medium for dissolving the antibodies
upon administration.
[0283] As shown in Example 11, 0304 or KMTR1 antibodies alone can
show cell-death-inducing activity. Moreover, as shown in this
example, 0304 was confirmed to have a significant anti-tumor effect
in the tumor-bearing mouse model. Hence, antibodies that can alone
show cell-death-inducing activity and anti-tumor activity are
expected to be able to show anti-tumor activity without depending
on the physiological conditions (e.g., types or numbers of
immunocytes) of a patient to which a prophylactic or therapeutic
agent against disease caused by TRAIL-R1 and/or TRAIL-R2-expressing
cells is to be administered, particular when a therapeutic agent
against malignant tumor is to be administered.
Example 13
Binding Affinity of Purified Human Anti-TRAIL-R1 and TRAIL-R2
Monoclonal Antibodies to TRAIL-R1 and TRAIL-R2
[0284] The binding affinity of purified human anti-TRAIL-R
monoclonal antibodies obtained in Example 10 to TRAIL-R was studied
by the following method using BIACORE 2000 (Biacore).
[0285] 1) Immobilization of TRAIL-R1 -hFc and TRAIL-R2-hFc
[0286] TRAIL-R1-hFc or TRAIL-R2-hFc was diluted at a final
concentration of 10 .mu.g/ml with 10 mM Acetic Acid (pH 4.0), and
then immobilized on a sensor chip CM5 by the amine coupling method.
The immobilization conditions are as follows. NHS activation and
ethanolamine blocking were performed according to the methods
described in the instructions. Coupling of TRAIL-R1-hFc and that of
TRAIL-R2-hFc were performed by manual injection as described in the
instructions.
[0287] (Immobilization conditions) Flow rate: 5 .mu.l/minute
[0288] NHS activation: 7 minutes
[0289] Coupling: manual injection
[0290] Ethanolamine blocking: 7 minutes
[0291] It was confirmed that 377.4 RU of TRAIL-R1-hFc and 495.4RU
of TRAIL-R2-hFc were immobilized on the sensor chip under the above
conditions.
[0292] 2) Regeneration Conditions and Confirmation of
Reproducibility
[0293] 20 .mu.g/ml purified human anti-TRAIL-R1 monoclonal antibody
2-6 was added for 2 minutes onto the sensor chip on which
TRAIL-R1-hFc had been immobilized. And then, the binding of the
antibodies to TRAIL-R1-hFc was confirmed. Subsequently, 50 mM NaOH
was added for 15 seconds, and then the complete dissociation of the
bound antibodies from TRAIL-R1-hFc was confirmed (hereinafter
complete dissociation is referred to as "regeneration"). Next, the
purified human anti-TRAIL-R1 monoclonal antibody 2-6 was added to
the regenerated TRAIL-R1-hFc at a flow rate of 20 .mu.l/minute by
the KINJECT method (binding for 1 minute and dissociation for 1
minute), followed by the addition of 50 mM NaOH for 15 seconds to
regenerate TRAIL-R1 hFc. This cycle was repeated 9 times. Even
after 9 repetitions of the above cycle, no change was found in the
amount of TRAIL-R1-hFc immobilized on the sensor chip or the amount
of the antibody bound thereon. It was thus revealed that
TRAIL-R1-hFc was regenerated without being inactivated by the
addition of 50 mM NaOH for 15 seconds. A similar examination was
performed using a sensor chip with TRAIL-R2-hFc immobilized thereto
and 20 .mu.g/ml purified human anti-TRAIL-R2 monoclonal antibody
E-11-13. As a result, it was confirmed that TRAIL-R2-hFc can be
regenerated under the same regeneration conditions.
[0294] 3) Examination of Interaction
[0295] Each of the purified human anti-TRAIL-R1 monoclonal
antibodies 1-13, 2-6 and 2-12 was serially diluted to 2.1, 4.2,
8.4, 16.8, 33.5, 67.0 and 134.0 nM using HBS-EP (Biacore). Each
antibody of the dilution series was added in order at a flow rate
of 20 .mu.l/minute by the KINJECT method (binding for 2 minutes and
dissociation for 6 minutes), thereby obtaining a sensorgram.
Similarly, each of the purified human anti-TRAIL-R2 monoclonal
antibodies E-11-13, L-30-10, H-48-2, F-4-8, W-40-6 and X-14-4 was
serially diluted to 0.52, 1.05, 2.1, 2.09, 4.19 and 8.38 nM using
HBS-EP (Biacore). Each antibody of the dilution series was added in
order at a flow rate of 20 .mu.l/minute by the KINJECT method
(binding for 2 minutes and dissociation for 2 minutes), thereby
obtaining sensorgrams. For each antibody, kinetics analysis was
performed using each sensorgram and BIAevaluation software ver3.2
(Biacore). As a fitting model, global fitting was performed using a
Bivalent model, so that the binding rate constant and the
dissociation rate constant were found. In addition, a dissociation
constant (Kd value) was calculated from the two constants. Also,
the sensorgrams were used for fitting after the subtraction of
control cells and buffer correction. Table 6 and Table 7 show the
results. In the tables, "kass" indicates the binding rate constant,
"kdiss" indicates the dissociation rate constant, and "K.sub.D"
indicates the dissociation constant.
7 TABLE 6 Purified human anti-TRAIL-R1 antibody kass (1/Ms) kdiss
(1/s) K.sub.D (nM) 1-13 1.08 .times. 10.sup.5 4.58 .times.
10.sup.-4 4.24 2-6 1.62 .times. 10.sup.5 1.86 .times. 10.sup.-4
1.15 2-12 1.63 .times. 10.sup.5 7.80 .times. 10.sup.-4 4.79
[0296]
8 TABLE 7 Purified human anti-TRAIL-R2 antibody kass (1/Ms) kdiss
(1/s) K.sub.D (nM) E-11-13 5.27 .times. 10.sup.5 3.84 .times.
10.sup.-5 0.0729 L-30-10 6.13 .times. 10.sup.5 1.44 .times.
10.sup.-3 2.35 H-48-2 5.75 .times. 10.sup.5 1.58 .times. 10.sup.-3
2.75 F-4-8 5.63 .times. 10.sup.5 7.05 .times. 10.sup.-4 1.25 W-40-6
1.74 .times. 10.sup.5 2.92 .times. 10.sup.-3 16.8 X-14-4 6.55
.times. 10.sup.4 2.93 .times. 10.sup.-3 44.7
Example 14
Preparation of Genes Encoding Monoclonal Antibodies and
Construction of Recombinant Antibody Expression Vectors
[0297] (1) cDNA Cloning and Construction of Expression Vectors of
E-11-13, L-30-10 and H-48-2 Antibody Genes
[0298] Hybridomas E-11-13, L-30-10 and H-48-2 were cultured in eRDF
media (Kyokutoseiyaku) containing 10 ng/ml Recombinant Human IL-6
(R&D Systems) and 10% Low IgG Fetal Bovine Serum (HyClone).
After the cells were collected by centrifugation, TRIZOL (Gibco
BRL) was added, and then total RNA was extracted according to the
instructions. Cloning of the variable regions of the antibody cDNAs
was performed using a SMART RACE cDNA amplification Kit (Clontech)
according to the attached instructions. Using 5 .mu.g of total RNA
as a template, 1st strand cDNA was prepared.
[0299] 1) Synthesis of 1st Strand cDNA
[0300] Total RNA 5 .mu.g/3 .mu.l
[0301] 5'CDS 1 .mu.l
[0302] SMART oligo 1 .mu.l
[0303] After the reaction solution having the above composition was
incubated at 70.degree. C. for 2 minutes,
[0304] 5.times. Buffer 2 .mu.l
[0305] DTT 1 .mu.l
[0306] DNTP mix 1 .mu.l and
[0307] Superscript II 1 .mu.l
[0308] were added, followed by incubation at 42.degree. C. for 1.5
hours.
[0309] Furthermore, after 100 .mu.l of Tricine buffer was added,
incubation was performed at 72.degree. C. for 7 minutes, thereby
obtaining 1st strand cDNA.
[0310] 2) Amplification by PCR of Heavy Chain Genes and Light Chain
Genes, and Construction of Recombinant Antibody Expression
Vector.
[0311] For cDNA amplification, Z-Taq (Takara) was used.
[0312] cDNA 2 .mu.l
[0313] 10.times.Z-Taq Buffer 5 .mu.l
[0314] dNTP mix 4 .mu.l
[0315] Z-Taq 1 .mu.l
[0316] Primer 1
[0317] Primer 2
[0318] A reaction solution having the above composition was
prepared to have a final volume of 50 .mu.l with double distilled
water, and then subjected to PCR.
[0319] To amplify heavy chains, UMP (SMART RACE cDNA amplification
Kit; Clontech) and hh-6 primer (5'-GGT CCG GGA GAT CAT GAG GGT GTC
CTT-3') (SEQ ID NO: 7) were used, and a cycle of 98.degree. C. for
1 second and 68.degree. C. for 30 seconds was repeated 30 times.
Furthermore, using 1 .mu.l of the reaction solution as a template,
NUMP (SMART RACE cDNA amplification Kit; Clontech) and hh-3 primer
(5'-GTG CAC GCC GCT GGT CAG GGC GCC TG-3') (SEQ ID NO: 8), a cycle
of 98.degree. C. for 1 second and 68.degree. C. for 30 seconds was
repeated 20 times. Subsequently, the amplified PCR product was
purified using a PCR purification kit (QIAGEN), and then the
nucleotide sequences were determined using hh-4 (5'-GGT GCC AGG GGG
AAG ACC GAT GG-3') (SEQ ID NO: 9) as a primer. Based on the
sequence information, it was found that the 3 clones of E-11-13,
L-30-10 and H-48-2 were identical in the sequence of the N-terminal
region. Thus, common primers were used for subcloning and the
determination of the nucleotide sequences. Based on the sequence
information, tnH48KBgl (5'-ATA TAG ATC TCT CAG TTA GGA CCC AGA GGG
AAC C-3') (SEQ ID NO: 10) was synthesized. Using this primer, the
sequences were also determined from the opposite direction. PCR was
performed using a specific primer and tnCHNhe (5'-GAT GGG CCC TTG
GTG CTA GCT GAG GAG ACG G-3') (SEQ ID NO: 11) (98.degree. C. for 1
second, 60.degree. C. for 30 seconds and 72.degree. C. for 30
seconds). The amplified heavy chain cDNA fragment was digested with
Sal I and Nhe I, and then introduced into an N5KG1-Val Lark vector
(an altered vector of IDEC Pharmaceuticals, N5KG1 (U.S. Pat. No.
6,001,358)) that had been cleaved using the same enzymes.
Sequencing was performed using the vector as a template so that the
inserted sequence was confirmed to be identical to the sequence
determined by a direct sequence.
[0320] Light chains were amplified by repeating a cycle of
98.degree. C. for 1 second and 68.degree. C. for 30 seconds 30
times using UMP (SMART RACE cDNA amplification Kit; Clontech) and
hk-2 primer (5'-GTT GAA GCT CTT TGT GAC GGG CGA GC-3') (SEQ ID NO:
12). Furthermore, using 1 .mu.l of the reaction solution as a
template, NUMP (SMART RACE cDNA amplification Kit; Clontech) and
hk-6 (5'-T GGC GGG AAG ATG AAG ACA GAT GGT G-3') (SEQ ID NO: 13), a
cycle of 98.degree. C. for 1 second and 68.degree. C. for 30
seconds was repeated 20 times. Subsequently, the amplified PCR
product was purified using a PCR purification kit (QIAGEN), and
then the nucleotide sequences were determined using hk-6 (5'-tggc
ggg aag atg aag aca gat ggt g-3') primer. Based on the sequence
information, it was found that 3 clones were all identical in the
sequence of the N-terminal region. Thus, common primers were used
for subcloning. Based on the sequence information, tnH48Hsal
(5'-ATA TGT CGA CTA CGG GGG GGC TTT CTG AGA GTC-3') (SEQ ID NO: 14)
was synthesized. Using this primer, sequencing was also performed
from the opposite direction. PCR was performed using a specific
primer and tnCkBsi (5'-AAG ACA GAT GGT GCA GCC ACC GTA CGT TTG
AT-3') (SEQ ID NO: 15) (98.degree. C. for 1 second, 60.degree. C.
for 30 seconds and 72.degree. C. for 30 seconds). The amplified
light chain cDNA fragment was digested with Bgl II and BsiW I, and
then introduced into a N5KG1-Val Lark vector that had been cleaved
with the same enzymes. Sequencing was performed using the vector as
a template so that the inserted sequence was confirmed to be
identical to the sequence determined by a direct sequence.
[0321] DNAs encoding the E-11-13 heavy chain variable region and
light chain variable region, and the amino acid sequences of the
heavy chain variable region and the light chain variable region,
are as respectively shown below.
9 <E-11-13 heavy chain variable region> (SEQ ID NO: 16)
GTCGACTACGGGGGGGCTTTCTGAGAGTCATGGATCTCATGTGCAAGAA
AATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGG
GTCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAG
CCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAT
CAGTAAAAGTTCCTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGG
GCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGTACCTTCTACAACC
CGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCA
GTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTAT
TACTGTGCGAGACTGACAGTGGCTGAGTTTGACTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCTAGC <E-11-13 heavy chain variable
region> (SEQ ID NO: 17)
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTV
SGGSIISKSSYWGWIRQPPGKGLEWIGSIYYSGSTFYNPSLKSRVTISVDTSK
NQFSLKLSSVTAADTAVYYCARLTVAEFDYWGQGTLVTVSSAS <E-11-13 light chain
variable region> (SEQ ID NO: 18)
TCACAGATCTCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGC
TCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAA
ATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA
GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGC
CTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGAT
GCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATT
TTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGC
CCTGGGACCAAAGTGGATATCAAACGTACG <E-11-13 light chain variable
region> (SEQ ID NO: 19)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSFL
AWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAV
YYCQQRSNWPLTFGPGTKVDIKRT
[0322] The translation initiation point of the heavy chain DNA is
an ATG codon that begins from the 30th adenine (A) from the 5' end
of SEQ ID NO: 16. The boundary of the antibody variable region and
the constant region is located between the 461st adenine (A) and
the 462nd guanine (G) from the 5'end. In the amino acid sequence,
the heavy chain variable region ranges from the N-terminus to the
144th serine (S) residue of SEQ ID NO: 17, and the constant region
is of the 145th alanine (A) and the following residues. Analysis of
the N-terminus of the purified heavy chain protein revealed that
the heavy chain signal sequence ranges from the N-terminus to the
26th serine (S) of SEQ ID NO: 17, and the N-terminus of the mature
protein is the 27th glutamine (Q) of SEQ ID NO: 17.
[0323] The translation initiation point of the light chain DNA is
an ATG codon that begins from the 35th A from the 5' end of SEQ ID
NO: 18, and the variable region ranges from the 5' end to the 415th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 127th lysine (K) of SEQ ID NO: 19.
Analysis of the N-terminus of the purified light chain protein
revealed that the light chain signal sequence ranges from the
N-terminus to the 20th glycine (G) of SEQ ID NO: 19, and the
N-terminus of the mature protein is the 21st glutamic acid (E) of
SEQ ID NO: 19.
[0324] DNAs encoding the L-30-10 heavy chain variable region and
light chain variable region and the amino acid sequences of the
heavy chain variable region and the light chain variable region,
are respectively shown below.
10 <L-30-10 heavy chain variable region> (SEQ ID NO: 20)
GTCGACTACGGGGGGGCTTTCTGAGAGTCATGGATCTCATGTGCAAGAA
AATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGG
GTCCTGTCCCAGTTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAG
CCCTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCA
GCAGTAGGAGTAACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGG
GGCTGGAGTGGATTGGGAATGTCTATTATAGAGGGAGCACCTACTACAA
TTCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAAC
CAGTTCTCCCTGAAGCTGAGCTCTGTGACCGTCGCAGACACGGCTGTGT
ATTACTGTGCGAGACTGTCAGTGGCTGAGTTTGACTACTGGGGCCAGGG
AATCCTGGTCACCGTCTCCTCAGCTAGC <L-30-10 heavy chain variable
region> (SEQ ID NO: 21)
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTV
SGGSISSRSNYWGWIRQPPGKGLEWIGNVYYRGSTYYNSSLKSRVTISVDT
SKNQFSLKLSSVTVADTAVYYCARLSVAEFDYWGQGILVTVSSAS <L-30-10 light
chain variable region> (SEQ ID NO: 22)
AGATCTCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAG
CTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGT
GTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGC
CACCCTCTCTTGTAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGG
TACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCAT
CCAACAGGGCCACTGGCAGCCCAGCCAGGTTCAGTGGCAGTGGGTCTG
GGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGC
AGTTTATTACTGTCAGCAGCGTAGCGACTGGCCTCTCACTTTCGGCCCT
GGGACCAAAGTGGATATCAAACGTACG <L-30-10 light chain variable
region> (SEQ ID NO: 23)
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQSVSSFL
AWYQQKPGQAPRLLIYDASNRATGSPARFSGSGSGTDFTLTISSLEPEDFAV
YYCQQRSDWPLTFGPGTKVDIKRT
[0325] The translation initiation point of the heavy chain DNA is
an ATG codon that begins from the 30th adenine (A) from the 5' end
of SEQ ID NO: 20. The boundary of the antibody variable region and
the constant region is located between the 461st adenine (A) and
the 462nd guanine (G) from the 5'end. In the amino acid sequence,
the heavy chain variable region ranges from the N-terminus to the
144th serine (S) residue of SEQ ID NO: 21, and the constant region
is of the 145th alanine (A) and the following residues. It was
predicted by a gene sequence prediction software (Signal P ver.2)
that the heavy chain signal sequence ranges from the N-terminus to
the 26th serine (S) of SEQ ID NO: 21. Analysis of the N-terminus of
the purified heavy chain protein revealed that the heavy chain
signal sequence ranges from the N-terminus to the 26th serine (S)
of SEQ ID NO: 21, and the N-terminus of the mature protein is the
27th glutamine (Q) of SEQ ID NO: 21.
[0326] The translation initiation point of the light chain DNA is
an ATG codon that begins from the 31st A from the 5' end of SEQ ID
NO: 22, and the variable region ranges from the 5' end to the 411st
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 127th lysine (K) of SEQ ID NO: 23.
Analysis of the N-terminus of the purified light chain protein
revealed that the light chain signal sequence ranges from the
N-terminus to the 20th glycine (G) of SEQ ID NO: 23, and the
N-terminus of the mature protein is the 21st glutamic acid (E) of
SEQ ID NO: 23.
[0327] DNAs encoding the H-48-2 heavy chain variable region and
light chain variable region, and the amino acid sequences of the
heavy chain variable region and the light chain variable region,
are respectively shown below.
11 <H-48-2 heavy chain variable region> (SEQ ID NO: 24)
TCGACTACGGGGGGGCTTTCTGAGAGTCATGGATCTCATGTGCAAGAAA
ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGG
TCCTGTCCCAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGC
CTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAG
CAGTAGTAGTTACTACTGGGGCTGGGTCCGCCAGCCCCCAGGGAAGGG
GCTGGAGTGGATTGGGAGTATCCATTATAGTGGGAGTACTTTCTACAACC
CGTCCCTCAAGAGTCGAGTCACCATTTCCGTAGACACGTCCAAGAACCA
GTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGACTGTGTAT
TACTGTGCGAGACAGGGGTCTACTGTGGTTCGGGGAGTTTACTACTACG
GTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTA GC <H-48-2
heavy chain variable region> (SEQ ID NO: 25)
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLVKPSETLSLTCTV
SGGSISSSSYYWGWVRQPPGKGLEWIGSIHYSGSTFYNPSLKSRVTISVDTS
KNQFSLKLSSVTAADTTVYYCARQGSTVVRGVYYYGMDVWGQGTTVTV SSAS <H-48-2
light chain variable region> (SEQ ID NO: 26)
AGATCTCTCAGTTAGGACCCAGAGGGAACCATGGAAACCCCAGC- GCAG
CTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAATTGT
GTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGC
CACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCC
TGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTG
CATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGT
CTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTT
TGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCTGTACACTTTTG
GCCAGGGGACCAAGCTGGAGATCAAACGTACG <H-48-2 light chain variable
region> (SEQ ID NO: 27)
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRASQSVSSS
YLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYGSSPLYTFGQGTKLEIKRT
[0328] The translation initiation point of the heavy chain DNA is
an ATG codon that begins from the 29th adenine (A) from the 5' end
of SEQ ID NO: 24. The boundary of the antibody variable region and
the constant region is located between the 484th adenine (A) and
the 485th guanine (G) from the 5'end. In the amino acid sequence,
the heavy chain variable region ranges from the N-terminus to the
152nd serine (S) residue of SEQ ID NO: 25, and the constant region
is of the 153rd alanine (A) and the following residues. It was
predicted by a gene sequence prediction software (Signal P ver.2)
that the heavy chain signal sequence ranges from the N-terminus to
the 26th serine (S) of SEQ ID NO: 25. Analysis of the N-terminus of
the purified heavy chain protein revealed that the heavy chain
signal sequence ranges from the N-terminus to the 26th serine (S)
of SEQ ID NO: 25, and the N-terminus of the mature protein is the
27th glutamine (Q) of SEQ ID NO: 25.
[0329] The translation initiation point of the light chain DNA is
an ATG codon that begins from the 31 st A from the 5' end of SEQ ID
NO: 26, and the variable region ranges from the 5' end to the 417th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 129th lysine (K) of SEQ ID NO: 27.
Analysis of the N-terminus of the purified light chain protein
revealed that the light chain signal sequence ranges from the
N-terminus to the 20th glycine (G) of SEQ ID NO: 27, and the
N-terminus of the mature protein is the 21st glutamic acid (E) of
SEQ ID NO: 27.
[0330] (2) cDNA Cloning and Construction of Expression Vector of
the 0304 Antibody Gene
[0331] Hybridoma 0304 cells were collected by centrifugation, and
then approximately 900 .mu.g of RNA was purified according to
protocols using an ISOGEN RNA extraction reagent (NIPPON GENE).
Next, 13 .mu.g of polyA.sup.+RNA was obtained from 300 .mu.g of RNA
using OligoteX.TM.-dT30<Super>(TAKARA SHUZO CO., LTD.). A
cloning experiment was performed using a SMART RACE cDNA
Amplification Kit (Clontech company) according the attached
instructions using the obtained polyA.sup.+RNA as a material,
thereby obtaining the cDNA of the variable regions of the antibody
gene. Specifically, a first strand cDNA was synthesized by reverse
transcriptase using 1.0 .mu.g of the purified polyA.sup.+RNA as a
material. The H-chain leader sequence and variable region
(hereinafter also referred to as "HV") and the L-chain leader
sequence and variable region (hereinafter, also referred to as
"LV") were amplified by PCR using the obtained cDNA as a template
and a primer set: primers (for H-chain: IgG1p; for L-chain: hk-2)
for PCR specific to each of the DNAs of a human antibody heavy
chain (hereinafter, the heavy chain is also referred to as
"H-chain") constant region and light chain (hereinafter, the light
chain is also referred to as "L-chain") constant region, and an UMP
primer (an oligonucleotide complementary to the common sequence
prepared at the 5' end of the synthesized cDNA) attached to a SMART
RACE cDNA Amplification Kit. In the PCR, TaKaRa LA Taq.TM. (TAKARA
SHUZO CO., LTD.), which was Taq DNA Polymerase for LA PCR, was
used. The template DNA was added to a solution containing
1.times.LA PCR Buffer II (Mg.sup.2+ plus) and 400 .mu.M each of
dNTP Mixture (in final concentration), 0.2 .mu.M each of two types
of primers and 2.5 U TaKaRa LA Taq/50 .mu.l. Reaction was performed
by touchdown PCR (94.degree. C. for 5 seconds and 72.degree. C. for
3 minutes (5 cycles) o 94.degree. C. for 5 seconds, 70.degree. C.
for 10 seconds and 72.degree. C. for 3 minutes (5 cycles) o
94.degree. C. for 5 seconds, 68.degree. C. for 10 seconds and
72.degree. C. for 3 minutes (20 cycles)). The amplified PCR
fragments were collected by ethanol precipitation, collected by
agarose gel electrophoresis, and then purified using a QIAquick Gel
Extraction Kit (QIAGEN) which was a DNA purification kit using
membranes. For the purified HV and LV fragments, DNA nucleotide
sequences were determined using an ABI PRISM.RTM. 3700 DNA Analyzer
(Applied Biosystems). Furthermore, the amplified HV and LV
fragments were subcloned respectively into pGEM.RTM.-T Easy Vector
Systems (Promega) using the TA cloning method. For the plasmid DNAs
of the thus obtained clones, the nucleotide sequences of the insert
DNAs were analyzed. The results were compared with the results of
the direct sequence analysis made for the PCR product. The
sequences of the primers (H-chain: hh-4; L-chain: hk-5 and hk-6;
for pGEM.RTM. -T Easy Vector: SP6 and T7) used for the
determination of the DNA nucleotide sequences are shown in Table 8.
The results of the direct sequence analysis made for each of the HV
and LV PCR fragments and the results of the analysis of the DNA
nucleotide sequences of (subcloned) multiple clones were identical
precisely.
[0332] The 0304 L-chain leader sequence and variable region were
amplified by PCR using the DNA of the 0304 antibody L-chain as a
template, and primers that had been designed to add a restriction
enzyme site to the end for ligation. The sequences of the primer
set used herein are shown in Table 8 (C23LBCL and C23LBsi). The
obtained PCR fragments were collected by ethanol precipitation,
digested with a restriction enzyme Bgl II and then cleaved with
BsiW I. The digested product was subjected to agarose gel
electrophoresis, so that a fragment of approximately 400 bp was
collected. Purification was performed using a QIAquick Gel
Extraction Kit (QIAGEN) which was a DNA purification kit using
membranes. In the meantime, N5KG4-Val Lark which was a vector (IDEC
Pharmaceuticals, an altered vector of N5KG1 (U.S. Pat. No.
6,001,358)) was similarly digested with restriction enzymes Bgl II
and BsiW I sequentially, and then subjected to dephosphorylation
treatment (treated with Alkaline Phosphatase (E. coli C75) (TAKARA
SHUZO CO., LTD.)). Then, less than approximately 9 kb DNAs were
collected by agarose gel electrophoresis and a DNA purification
kit. These 2 fragments were ligated using T4 DNA ligase and then
introduced into Escherichia coli DH5.alpha., so as to obtain a
transformant. A plasmid DNA, N5KG4-0304L, that had been prepared by
inserting the 0304 antibody L-chain leader+variable region into
N5KG4-Val Lark was selected. The DNA nucleotide sequences
surrounding the inserted fragment were determined, thereby
confirming that there was no mutation or the like in the DNA
nucleotide sequences. To insert the H-chain variable region and the
like into the thus obtained N5KG4-0304L, this plasmid DNA was
sequentially cleaved with restriction enzymes Nhe I and Sal I,
dephosphorylation treatment was performed, and then an
approximately 9.3 kb vector DNA was purified. In the meantime, the
0304 antibody H-chain gene leader sequence and variable region were
amplified by PCR using the plasmid DNA of the antibody H-chain as a
template. The primer set used for amplification (T0304Sal and
T0304Nhe) is shown in Table 8.
[0333] The obtained PCR fragment was cleaved with restriction
enzymes Nhe I and Sal I, and then subjected to agarose gel
electrophoresis, thereby purifying approximately 450 bp fragments.
These 2 types of DNAs were ligated and introduced into Escherichia
coli to obtain transformants, and then clones having the target
H-chain leader sequence and variable region inserted therein were
selected. The DNA nucleotide sequence of the insertion portion was
determined, thereby confirming that there was no difference between
the inserted sequence amplified by PCR and the gene sequence used
as a template.
[0334] DNAs encoding the 0304 heavy chain variable region and light
chain variable region, and the amino acid sequences of the heavy
chain variable region and the light chain variable region, are
respectively shown below.
12 <0304 heavy chain variable region> (SEQ ID NO: 28)
CTCAACAACC ACATCTGTCC TCTAGAGAAA ACCCTGTGAG CACAGCTGCT CACCATGGAC
TGGAGCTGGA GGATCCTCTT CTTGGTGGCA GCAGCTACAA GTGCCCACTC CCAGGTGCAG
CTGGTGCAGT CTGGGGCTGA GATGAAGAAG CCTGGGGCCT CAGTCAAGGT GTCCTGCAAG
ACTTCTGGAT ACACCTTCAC GAATTATAAG ATCAACTGGG TGCGACAGGC CCGTGGACAA
GGACTTGAGT GGATGGGATG GATGAACCCT GACACTGATA GCACAGGCTA TCCACAGAAG
TTCCAGGGCA GAGTCACCAT GACCAGGAAC ACCTCCATAA GGAGAGCCTA CATGGAGCTG
AGCAGCCTGA GATCTGAGGA CACGGCCGTG TATTACTGTG CGAGATCCTA TGGTTCGGGG
AGTTATTATA GAGACTATTA CTAGGGTATG GACGTCTGGG GCCAAGGGAC CACGGTCACC
GTCTCCTCA <0304 heavy chain variable region> (SEQ ID NO: 29)
MDWTWRILFL VAAATSAHSQ VQLVQSGAEM KKPGASVKVS CKTSGYTFTN YKINWVRQAP
GQGLEWMGWM NPDTDSTGYP QKFQGRVTMT RNTSISTAYM ELSSLRSEDT AVYYCARSYG
SGSYYRDYYY GMDVWGQGTT VTVSS <0304 light chain variable
region> (SEQ ID NO: 30) GAGGAACTGC TCAGTTAGGA CCCAGAGGGA
ACCATGGAAG CCCCAGCTCA GCTTCTCTTC CTCCTGCTAC TCTGGCTCCC AGATACCACC
GGAGAAATTG TGTTGACACA GTCTCCAGCC ACCCTGTCTT TGTCTCGAGG GGAAAGAGCC
ACCCTCTCCT GCAGGGCCAG TCAGAGTGTT AGCAGCTACT TAGCCTGGTA CCAACAGAAA
CCTGGCCAGG CTCCCAGGGT CCTCATCTAT GATGGATCCA ACAGGGCCAC TGGCATCCCA
GCCAGGTTCA GTGGCAGTGG GTCTGGGAGA GACTTCACTC TCAGCATCAG CAGCCTAGAG
CCTGAAGATT TTGCAGTTTA TTACTGTGAG GAGCGTAGCA ACTGGCCGCT CAGTTTCGGC
GGAGGGACCA AGGTGGAGAT CAAACGA <0304 light chain variable
region> (SEQ ID NO: 31) MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT
LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD
FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG GTKVEIKR
[0335] The translation initiation point of the heavy chain DNA is
an ATG codon that begins from the 55th adenine (A) from the 5' end
of SEQ ID NO: 28. The antibody variable region ranges from the 5'
end to the 489th adenine (A). In the amino acid sequence, the heavy
chain variable region ranges from the N-terminus to the 145th
serine (S) residue of SEQ ID NO: 29. It was predicted by a gene
sequence prediction software (Signal P ver.2) that the heavy chain
signal sequence ranges from the N-terminus to the 19th serine (S)
of SEQ ID NO: 29. The N-terminus of the mature protein is thought
to be the 20th glutamine (Q) of SEQ ID NO: 29.
[0336] The translation initiation point of the light chain DNA is
an ATG codon that begins from the 34th A from the 5' end of SEQ ID
NO: 30, and the variable region ranges from the 5' end to the 414th
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 127th lysine (K) of SEQ ID NO: 31. It
was predicted by a gene sequence prediction software (Signal P
ver.2) that the light chain signal sequence ranges from the
N-terminus to the 20th glycine (G) of SEQ ID NO: 31. The N-terminus
of the mature protein is thought to be the 21st glutamic acid (E)
of SEQ ID NO: 31.
13TABLE 8 Nucleotide Sequence of Synthetic DNA Primer SEQ ID No
name Sequence (5' to 3') Length NO: 1 IgG1
TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG 31-mer 36 2 hk-2
GTTGAAGCTCTTTGTGAGGGGCGAGC 26-mer 12 3 hh-4 GGTGCCAGGGGGAAGACCGATGG
23-mer 9 4 hk-5 AGGCACACAACAGAGGCAGTTCCAGATTTC 30-mer 37 5 hk-6
TGGCGGGAAGATGAAGACAGATGGTG 26-mer 13 6 SP6 GATTTAGGTGACACTATAG
19-mer 38 7 T7 TAATACGACTCACTATAGGG 20-mer 39 8 C23LBCL
ATCACAGATCTCTCACCATGGAAGCCCCAGCTCAGCT- TCTC 41-mer 40 9 C23LBsi
GGTGCAGCCACCGTACGTTTGATCTCCACCTTG 33-mer 41 10 T0304Sal
GCGACTAAGTCGACACCATGGACTGGACCTGGAGG- ATC 38-mer 42 11 T0304Nhe
AGAGAGAGAGGCTAGCTGAGGAGACGGTGACC 32-mer 43 12 SEQU1783
GGTACGTGAACCGTCAGATCGCCTGGA 27-mer 44 13 SEQU4618
TCTATATAAGCAGAGCTGGGTACGTCC 27-mer 45
[0337] (3) cDNA Cloning of KMTR1 Antibody Gene
[0338] Hybridoma KMTR1 cells were collected by centrifugation, and
then approximately 900 .mu.g of RNA was purified using an ISOGEN
RNA extraction reagent (NIPPON GENE) according to protocols. Next,
13 .mu.g of polyA.sup.+RNA was obtained from 300 .mu.g of RNA using
Oligotex.TM.-dT30<Super>(TAKARA SHUZO CO., LTD.). A cloning
experiment was performed using a SMART RACE cDNA Amplification Kit
(Clontech company) according the attached instructions using the
obtained polyA.sup.+RNA as a material, thereby obtaining the cDNA
of the variable regions of the antibody gene. Specifically, a first
strand cDNA was synthesized by reverse transcriptase using 1.0
.mu.g of the purified polyA.sup.+RNA as a material. The H-chain
leader sequence and variable region (hereinafter also referred to
as "HV") and the L-chain leader sequence and variable region
(hereinafter, also referred to as "LV") were amplified by PCR using
the obtained cDNA as a template and a primer set: primers (for
H-chain: IgG1p; for L-chain: hk-2) for PCR specific to each of the
DNAs of a human antibody heavy chain (hereinafter, the heavy chain
is also referred to as "H-chain") constant region and light chain
(hereinafter, the light chain is also referred to as "L-chain")
constant region, and an UMP primer (an oligonucleotide
complementary to the common sequence prepared at the 5' end of the
synthesized cDNA) attached to a SMART RACE cDNA Amplification Kit.
In the PCR, TaKaRa LA Taq.TM. (TAKARA SHUZO CO., LTD.), which was
Taq DNA Polymerase for LA PCR, was used. The template DNA was added
to a solution containing 1.times.LA PCR Buffer II (Mg.sup.2+ plus)
and 400 .mu.M each of dNTP Mixture (in final concentration), 0.2
.mu.M each of two types of primers and 2.5 U TaKaRa LA Taq/50
.mu.l. Reaction was performed by touchdown PCR (94.degree. C. for 5
seconds and 72.degree. C. for 3 minutes (5
cycles).fwdarw.94.degree- . C. for 5 seconds, 70.degree. C. for 10
seconds and 72.degree. C. for 3 minutes (5
cycles).fwdarw.94.degree. C. for 5 seconds, 68.degree. C. for 10
seconds and 72.degree. C. for 3 minutes (20 cycles)). The amplified
PCR fragment was collected by ethanol precipitation, collected by
agarose gel electrophoresis, and then purified using a QIAquick Gel
Extraction Kit (QIAGEN) which was a DNA purification kit using
membranes. For the purified HV and LV fragments, DNA nucleotide
sequences were determined using an ABI PRISM.RTM. 3700 DNA Analyzer
(Applied Biosystems). Furthermore, the amplified HV and LV
fragments were subcloned respectively into pGEM.RTM.-T Easy Vector
System (Promega) using the TA cloning method. For the plasmid DNAs
of the thus obtained clones, the nucleotide sequences of the insert
DNAs were analyzed. The results were compared with the results of
the direct sequence analysis made for the PCR product. The
sequences of the primers used for the determination of the DNA
nucleotide sequences (H-chain: hh-4; L-chain: hk-5 and hk-6; for
pGEM.RTM.-T Easy Vector: SP6 and T7) are shown in Table 8 above.
The results of the direct sequence analysis made for each of the HV
and LV PCR fragments and the results of the analysis of the DNA
nucleotide sequences of multiple clones (subcloned) were identical
precisely. The thus determined DNA nucleotide sequences and the
amino acid sequences of the H-chain and the L-chain of the human
antibody gene expressed on the KMTR1 cell are shown.
14 <KMTR1 heavy chain variable region> (SEQ ID NO: 32)
GAGCTCTGAG AGAGGAGCCC AGCCCTGGGA TTTTCAGGTG TTTTCATTTG GTGATCAGGA
CTGAACAGAG AGAACTCACC ATGGAGTTTG GGCTGAGCTG GCTTTTTGTT GTGGCTATTT
TAAAAGGTGT CCAGTGTGAG GTAGAGCTGT TGGAGTCTGG GGGAGGCTTG GTAGAGCCTG
GGAGGTCCCT GAGACTCTCC TGTGCAGCCT CTGGATTCAC CTTTAGCAGC TATGCCATGA
GCTGGGTCCG CCAGGCTCCA GGGAAGGGGC TGGAGTGGGT CTCAGCTATT AGTGGTAGTG
GTGGTAGGAG ATACTACGCA GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT
CCAAGAACAC GCTGTATCTG CAAATGAAGA GCCTGAGAGC CGAGGACACG GCCGTATATT
ACTGTGCGAA AGAGAGCAGT GGCTGGTTCG GGGGGTTTGA CTACTGGGGC CAGGGAACGC
TGGTCACCGT CTCCTCA <KMTR1 heavy chain variable region> (SEQ
ID NO: 33) MEFGLSWLFL VAILKGVQCE VQLLESGGGL VQPGRSLRLS CAASGFTFSS
YAMSWVRQAP GKGLEWVSAI SGSGGSRYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT
AVYYCAKESS GWFGAFDYWG QGTLVTVSS <KMTR1 light chain variable
region> (SEQ ID NO: 34) GATCTTAAAA GAGGTTCTTT CTCTGGGATG
TGGCATGAGG AAAACTGACA AGTCAAGGCA GGAAGATGTC GCCATCACAA CTCATTGGGT
TTCTGCTGCT CTGGGTTCCA GCCTCCAGGG GTGAAATTGT GCTGACTCAG TCTCCAGACT
TTCAGTCTGT GACTCCAAAG GAGAAAGTCA CCATCACCTG CCGGGCCAGT CAGAGCATTG
GTAGTAGCTT ACACTGGTAC CAGCAGAAAC CAGATCAGTC TCCAAAGCTC CTCATCAAGT
ATGCTTCCGA GTCCTTCTCA GGGGTCCGCT GGAGGTTCAG TGGGAGTGGA TCTGGGACAG
ATTTCACCCT CACCATCAAT AGCCTGGAAG GTGAAGATGC TGCAGCGTAT TACTGTCATC
AGAGTAGTAG TTTACCGATC ACCTTCGGCC AAGGGACACG AGTGGAGATT AAAGGA
<KMTR1 light chain variable region> (SEQ ID NO: 35)
MSPSQLIGFL LLWVPASRGE IVLTQSPDFQ SVTPKEKVTI TCRASQSIGS SLHWYQQKPD
QSPKLLIKYA SQSFSGVPSR FSGSGSGTDF TLTINSLEAE DAAAYYCHQS SSLPITFGQG
TRLEIKR
[0339] The translation initiation point of the heavy chain DNA is
an ATG codon that begins from the 81st adenine (A) from the 5' end
of SEQ ID NO: 32. The antibody variable region ranges from the 5'
end to the 497th adenine (A). In the amino acid sequence, the heavy
chain variable region ranges from the N-terminus to the 139th
serine (S) residue of SEQ ID NO: 33. It was predicted by a gene
sequence prediction software (Signal P ver.2) that the heavy chain
signal sequence ranges from the N-terminus to the 19th cysteine (C)
of SEQ ID NO: 33. The N-terminus of the mature protein is thought
to be the 20th glutamic acid (E) of SEQ ID NO: 33.
[0340] The translation initiation point of the light chain DNA is
an ATG codon that begins from the 66th A from the 5' end of SEQ ID
NO: 34, and the variable region ranges from the 5' end to the 443rd
adenine (A). In the amino acid sequence, the variable region ranges
from the N-terminus to the 126th lysine (K) of SEQ ID NO: 35. It
was predicted by a gene sequence prediction software (Signal P
ver.2) that the light chain signal sequence ranges from the
N-terminus to the 19th glycine (G) of SEQ ID NO: 35. The N-terminus
of the mature protein is thought to be the 20th glutamic acid (E)
of SEQ ID NO: 35.
Example 15
Preparation of Recombinant Antibody
[0341] The recombinant antibody expression vector constructed in
Example 14 was introduced into a host cell, thereby preparing a
recombinant antibody-expressing cell. As the host cell for
expression, for example, a dhfr-deficient strain (ATCC CRL-9096) of
CHO cells was used. The vector was introduced into a host cell by
electroporation. Approximately 2 .mu.g of the antibody expression
vector was linearized with a restriction enzyme. Under conditions
of 350 V and 500 .mu.F, the gene was introduced into
4.times.10.sup.6 CHO cells using a Bio-Rad electrophoreter, and
then the cells were seeded in a 96-well culture plate. A drug
corresponding to a selection marker of the expression vector was
added, and then culturing was continued. After colonies were
confirmed, antibody-expressing lines were selected by the method
described in Example 4. The antibodies were purified from the
selected cells as described in Example 10.
Example 16
Cell-death-inducing Activity of Recombinant Antibody on Carcinoma
Cell
[0342] The cell-death-inducing activity on Colo205 (ATCC No.
CCL-222), the colon carcinoma cell, was measured using the
recombinant human anti-TRAIL-R2 monoclonal antibodies obtained in
Example 15. Colo205 cells cultured in RPMI media containing 10% FCS
were prepared at a concentration of 1.0.times.10.sup.5/ml, and then
100 .mu.l of the suspension was added to each well of a 96-well
flat-bottomed plate (Beckton Dickinson). After 24 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas, the
purified antibodies E11 (CHO-3) and H48 (CHO-3) were added (10
.mu.l/well) at final concentrations of 10, 100, 1000 and 10000
ng/ml. Further, 10 .mu.l of goat anti-human IgG (.gamma.)-specific
polyclonal antibodies (Sigma) were added to each well at final
concentrations of 10 and 100 .mu.g/ml. For the obtained hybridomas,
wells supplemented with no goat anti-human IgG (.gamma.)-specific
polyclonal antibodies were prepared. As a positive control, human
recombinant TRAIL proteins (R&D SYSTEMS) with final
concentrations of 1 and 10 ng/ml were used. A human anti-HSA
antibody was used as a negative control. After 48 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas, an MTS
reagent (Cell Titer 96 AQ.sub.UEOUS Non-Radioactive Cell
Proliferation Assay: Promega) was prepared according to the method
described in the instructions. 20 .mu.l of the reagent was added to
each well. After 2 hours of culturing at 37.degree. C. under 5.0%
carbon dioxide gas, absorbance at a wavelength of 490 nm (reference
wavelength of 630 nm) was measured using a microplate reader (1420
ARVO multi-label counter: WALLAC). Using the reducibility of the
mitochondria as an indicator, the survival rate of the cells was
calculated. The survival rate of the cells in each well was
calculated using a formula similar to that of Example 7.
[0343] FIG. 11a and 11b show the results. FIG. 11a shows the result
of an experiment wherein no goat anti-human IgG(.gamma.)-specific
polyclonal antibody was added, and FIG. 11b shows the result of an
experiment wherein goat anti-human IgG(.gamma.)-specific polyclonal
antibodies were added.
[0344] As shown in FIG. 11a, the recombinant antibodies E11(CHO-3)
and H48(CHO-3) have activity to induce cell death in Colo205 cells
in the case of the antibody alone. Moreover, as shown in FIG. 11b,
when goat anti-human IgG(.gamma.)-specific polyclonal antibodies
were added, the recombinant antibodies showed cell-death-inducing
activity equivalent to that of the antibody purified from the
culture supernatant of the hybridoma.
[0345] Anti TRAIL-R Antibody Which Induces Apoptosis Independently
of Exogenous Factors and as a Monomer of the Antibody
[0346] Preparation of antigen, generation of human
antibody-producing mice and preparation of human monoclonal
antibodies to human TRAIL-R2 were performed in the same procedure
as in Examples 1 to 3.
[0347] By the activity to induce cell death on carcinoma cells
described in Example 7, ELISA described in Examples 4 and 5 and the
FACS analysis described in Example 6, a large number of hybridomas
producing human monoclonal antibodies that have the activity to
induce cell death only by the hybridoma culture supernatant, and
have human immunoglobulin .gamma. chain (hIg .gamma.) and human
immunoglobulin light chain .kappa., and have reactivity
specifically to human TRAIL-R2 were obtained. Furthermore, in any
of the following examples including this example, and tables and
figures showing the test results of the examples, hybridoma clones
producing each of the human anti-human TRAIL-R2 monoclonal
antibodies of the present invention were denoted using symbols. A
clone represented by symbols with the term "antibody" placed before
or after the symbols means an antibody that is produced by each of
the hybridomas, or a recombinant antibody that is produced by a
host cell carrying an antibody gene (full-length or a variable
region) isolated from the hybridoma. In addition, within a
contextually clear range, the name of a hybridoma clone may express
the name of an antibody. The following hybridoma clones represent
single clones:0304 and 0322. 0304 was internationally deposited as
described above.
Example 17
Cell-death-inducing Activity on Carcinoma Cells
[0348] Using the culture supernatant of the hybridoma producing the
human anti-TRAIL-R2 monoclonal antibodies obtained from Example 3,
cell-death-inducing activity on Colo205 (ATCC No. CCL-222) cells,
which were colon carcinoma cells, was measured. Colo205 cells
cultured in RPMI-1640 medium containing 10% FCS were prepared at a
concentration of 1.0.times.10.sup.5/ml. 50 .mu.l of the suspension
was added to each well of a 96-well flat bottomed plate (Beckton
Dickinson). Then, 50 .mu.l of the culture supernatant of the
hybridomas was added to each well. The wells in which culture
medium for a hybridoma was added were prepared as a negative
control. After 24 hours of culturing at 37.degree. C. under 5.0%
carbon dioxide gas, an MTS reagent (Cell Titer 96.RTM. AQUEOUS
Non-Radioactive Cell Proliferation Assay: Promega) was prepared
according to the method described in the instructions, and then 20
.mu.l of the reagent was added to each well. After another 2 hours
of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
absorbance at a wavelength of 490 nm (reference wavelength of 630
nm) was measured using a microplate reader (1420 ARVO multi-label
counter: WALLAC). Using the reducibility of mitochondria as an
indicator, the survival rate of the cells was calculated. The
survival rate of the cells in each well was calculated by the
following formula: Survival rate (%)=100.times.(a-b)/(c-b) (wherein
"a" represents the measured value of a well tested, "b" represents
the measured value of a cell-free well, and "c" represents the
measured value of a well containing cells). Specifically, "c"
represents the measured value of a well containing a control
antibody which has the same subclass with the antibody bound to
TRAIL-R and is not bound to the carcinoma cells. Alternatively,
when an antibody is a whole antibody or a functional fragment
thereof, "c" represents (i) the measured value of a well containing
carcinoma cells and a control antibody which is not bound to
carcinoma cells and has the same subclass with the antibody or the
functional fragment thereof bound to TRAIL-R when the antibody or
the functional fragment thereof has a constant region, or (ii) the
measured value of a well containing carcinoma cells and a control
antibody which is not bound to the carcinoma cells and does not
have a constant region when the antibody or the functional fragment
thereof does not have a constant region. Table A-1 shows the
results. Table A-1 shows the cell-death-inducing activity (in the
culture supernatant of the hybridomas 0304 and 0322 producing the
human anti-TRAIL-R2 monoclonal antibodies) on Colo205.
15 TABLE A-1 Human anti-TRAIL-R2 antibody Colo205 cell survival
rate 0304 - 0322 - ++: Survival rate of 80% or more +: Survival
rate of 21% to 79% -: Survival rate of 20% or less
[0349] As a result, it was revealed that the human anti-TRAIL-R2
monoclonal antibody clearly has the activity to induce cell death
in Colo205 cells compared to a negative control in a state without
cross-linking wherein hybridoma culture supernatant only was
added.
Example 18
Detection of Human Anti-TRAIL-R2 Monoclonal Antibody Having Human
Immunoglobulin Light Chain .kappa. (Ig.kappa.)
[0350] Fusion proteins of the extracellular regions of human
TRAIL-R1 and R2 and the Fc region of human IgG1 (hereinafter
referred to as "TRAIL-R1-hFc" and "TRAIL-R2-hFc" (ALEXIS).
Regarding TRAIL-R2-hFc, a region comprising 1 to 183 amino acids
was also used as the extracellular region of TRAIL-R2) were added
at 0.5 .mu.g/ml in phosphate buffered saline (hereinafter referred
to as "PBS"). 50 .mu.l of the thus prepared solution was added to
each well of a 96-well microplate for ELISA (Maxisorp, Nunc) and
incubated for 1 hour at room temperature or at 4.degree. C.
overnight, thereby coating TRAIL-R1-hFc or TRAIL-R2-hFc to the
microplate. Subsequently the supernatant was discarded, a blocking
reagent (SuperBlock (registered trademark) Blocking Buffer, PIERCE)
was added to each well, and then incubation was performed at room
temperature for 30 minutes, thereby blocking the part where
TRAIL-R1-hFc or TRAIL-R2-hFc did not bind. Thus, a microplate
having each well coated with TRAIL-R1-hFc or TRAIL-R2-hFc was
prepared.
[0351] The culture supernatant of each hybridoma (50 .mu.l) was
added to each well, reaction was performed at room temperature for
1 hour, and then each well was washed three times in 0.1%
Tween20-containing PBS (PBS-T). Subsequently, horseradish
peroxidase-labeled goat anti-human Ig.kappa. antibodies (50
.mu.l/well, Biosource International) were diluted 2000 times in
PBS-T containing 10% Block Ace (Dainippon Pharmaceutical Co.,
Ltd.). 50 .mu.l of the thus prepared solution was added to each
well, and incubation was then performed at room temperature for 30
minutes; The microplate was washed three times with PBS-T, and then
50 .mu.l of a TMB chromogenic substrate solution (DAKO) was added
to each well, followed by incubation at room temperature for 20
minutes. 0.5M sulfuric acid was added (50 .mu.l/well) to each well
to stop reaction. Absorbance at a wavelength of 450 nm (reference
wavelength of 570 nm) was measured with a microplate reader (1420
ARVO multi-label counter: WALLAC). Table A-2 shows the
characteristics of the thus obtained anti-human TRAIL-R2. Table A-2
shows the subclass and cross reactivity of the obtained human
anti-TRAIL-R2 monoclonal antibodies 0304 and 0322.
16TABLE A-2 Human anti-TRAIL- Cross reactivity R2 antibody Subclass
TRAIL-R1 TRAIL-R2 0304 IgG4 - + 0322 IgG4 - + +: with reactivity -:
no reactivity
Example 19
Identification of the Subclass of Each Monoclonal Antibody
[0352] Rabbit anti-huma IgG antibody (DAKO) which was 500 times
diluted with 50 mM Sodium bicarbonate buffer was added into wells
of 96-well microplate for ELISA (Maxisorp, Nunc) at 50 .mu.l/well
and incubated at 4.degree. C. overnight to be coated to the
microplate. Subsequently, the supernatant was discarded, a blocking
reagent (SuperBlock (registered trademark) Blocking Buffer, PIERCE)
was added to each well, and then incubation was performed at room
temperature for 30 minutes to block the site to which rabbit
anti-human IgG antibody was not bound to. Thus, the microplates of
which each well was coated with rabbit anti-human IgG antibody were
prepared. The culture supernatant (50 .mu.l) of the hybridomas was
added to be reacted for 1 hour, and then each well was washed three
times with PBS-T. Subsequently, sheep anti-human IgG1 antibodies,
sheep anti-human IgG2 antibodies, or sheep anti-human IgG3
antibodies or sheep anti-human IgG4 antibodies, which had been
respectively labeled with horseradish peroxidase and diluted 2000
times, were added (50 .mu.l/well, The Binding Site) to each well,
followed by incubation at room temperature for 1 hour. After
washing 3 times with PBS-T, a substrate buffer (TMB, 50 .mu.l/well,
DAKO) was added to each well, and then incubation was performed at
room temperature for 20 minutes. Next, 0.5M sulfuric acid (50
.mu.l/well) was added to stop the reaction. Absorbance at a wave
length of 450 nm (with a reference wavelength of 570 nm) was
measured using a microplate reader (1420 ARVO multi-label counter:
WALLAC). The results are shown in Table A-2 above.
Example 20
Test of the Reactivity of Each Monoclonal Antibody to TRAIL-R1 and
R2 Expressing Cells
[0353] The reactivity of each of the monoclonal antibodies obtained
in Example 21 to the TRAIL-R1delta-expressing L929 cells and
TRAIL-R2delta-expressing L929 cells prepared in Example 1 was
examined by FACS analysis. L929 cells, TRAIL-R1delta-expressing
L929 cells and TRAIL-R2delta-expressing L929 cells were suspended
at a concentration of 2.times.10.sup.6/ml in a staining buffer (SB)
of PBS containing 2mM EDTA, 0.1% NaN.sub.3 and 1% FCS. The cell
suspension (100 .mu.l/well) was added into a 96-well round-bottomed
plate (Beckton Dickinson). After centrifugation (2000 rpm,
4.degree. C., 2 minutes), the supernatant was removed and then the
purified antibody obtained by the method described in Example 8 (1
.mu.g/ml, 100 .mu.l) was added. The mixture was agitated, allowed
to stand on ice for 30 minutes, and then subjected to
centrifugation (2000 rpm, 4.degree. C. for 2 minutes) to remove the
supernatant. After the pellet was washed three times with SB (100
.mu.l/well), RPE fluorescence-labeled rabbit anti-human Ig.kappa.
F(ab').sub.2 antibodies (DAKO) 10 times diluted with SB was added
at 100 .mu.l/well, and then incubation was performed on ice for 30
minutes. After washed three times with SB, the cells were suspended
in 300 .mu.l of SB, and then fluorescence intensity of each cell
was measured by FACS (FACScan, Beckton Dickinson). All the
antibodies 0304 and 0322 were confirmed to have strong binding
activity only to the TRAIL-R2delta-expressing L929 cells, and no
binding activity to L929 cells and TRAIL-R1delta-expressing L929
cells was observed. Thus, it was confirmed that they were
antibodies binding specifically to TRAIL-R2.
Example 21
Preparation of Each Antibody
[0354] The human anti-TRAIL-R2 monoclonal antibodies 0304 and 0322
were purified by the following method. The culture supernatant
containing the human anti-TRAIL-R2 monoclonal antibodies was
prepared by the following method. First, human anti-TRAIL-R2
monoclonal antibodies-producing hybridomas were adapted in an eRDF
medium (Kyokutoseiyaku) containing 10 ng/ml recombinant human IL-6
(R&D Systems) and 10% Low IgG fetal bovine serum (HyClone). The
adapted hybridomas were cryopreserved. Next, for the purpose of
antibody purification, a part of the hybridomas was adapted in an
eRDF medium (Kyokutoseiyaku) containing 1.times.
Insulin-Transferrin-Selenium-X Supplement (Gibco BRL), 10 ng/ml
recombinant human IL-6 (R&D Systems) and 1% Low IgG fetal
bovine serum (HyClone). The hybridoma cells were cultured in
flasks, and when the viable cell ratio of the hybridoma reached
90%, the culture supernatant was collected. The collected
supernatant was applied to a 10 .mu.m filter and a 0.2 .mu.m filter
(German Science), thereby removing miscellaneous waste materials
such as hybridomas.
[0355] The human anti-TRAIL-R2 monoclonal antibodies from the
culture supernatant as prepared above were purified by the
following method. The culture supernatant containing the human
anti-TRAIL-R2 monoclonal antibody was subjected to affinity
purification using rmp Protein A (Amersham Pharmacia Biotech), a
0.8.times.40 cm column (Bio-Rad), PBS as an adsorption buffer and a
0.02 M glycine buffer (pH 3) as an elution buffer. The eluted
fraction was adjusted to have a pH of around 7.2 by adding 1 M Tris
(pH 9.0). The thus prepared antibody solution was substituted with
PBS using a dialysis membrane (10000 cut, Spectrum Laboratories),
and then filtered using a MILLEX-GV membrane filter (Millipore)
with a pore size of 0.22 .mu.m for sterilization, thereby obtaining
purified human anti-TRAIL-R2 monoclonal antibody. Absorbance at 280
nm was measured, and then the concentration of the purified
antibodies was calculated using 1.4 OD=1 mg/ml.
Example 22
Cell-death-inducing Activity on Carcinoma Cells by the Purified
Human Anti-TRAIL-R2 Monoclonal Antibody
[0356] Using the purified human anti-TRAIL-R2 monoclonal antibody
obtained from Example 21, cell-death-inducing activity on Colo205
(ATCC No. CCL-222) cells, which were colon carcinoma cells, was
measured. Colo205 cells cultured in RPMI-1640 medium containing 10%
FCS were prepared at a concentration of 1.0.times.10.sup.5/ml. 100
.mu.l of the suspension was added to each well of a 96-well flat
bottomed plate (Beckton Dickinson). After culturing at 37.degree.
C. under 5.0% carbon dioxide gas for 24 hours, the purified
antibody (final concentration: 10, 30, 100, 300, 1000 ng/ml) was
added at 10 .mu.l/well. Furthermore, goat anti-human IgG
(.gamma.)-specific polyclonal antibodies (Sigma) were added (10
.mu.l/well) at a final concentration of 10 .mu.g/ml as a
cross-linker to each well. Wells not supplemented with goat
anti-human IgG (.gamma.)-specific polyclonal antibodies were
prepared. As a positive control, human recombinant TRAIL protein
(R&D SYSTEMS) was employed with a final concentration of 1, 3
and 10 ng/ml. As a negative control, human anti-DNP antibody was
employed. The antibody was subjected to react with the receptor on
the surface of the cells by culturing 48 hours at 37.degree. C.
under 5.0% carbon dioxide gas. The reaction volume for each
reaction system was 120 .mu.l. Then, each well was washed once with
PBS and added with fresh RPMI-1640 medium containing 10% FCS.
Subsequently, an MTS reagent (Cell Titer 96.RTM. AQUEOUS
Non-Radioactive Cell Proliferation Assay: Promega) was prepared
according to the method described in the instructions, and then 20
.mu.l of the reagent was added to each well. After another 1 hour
of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
absorbance at a wavelength of 490 nm (reference wavelength of 630
nm) was measured using a microplate reader (1420 ARVO multi-label
counter: WALLAC). Using the reducibility of mitochondria as an
indicator, the survival rate of the cells was calculated. The
survival rate of the cells in each well was calculated by the
following formula: Survival rate (%)=100.times.(a-b)/(c-b) (wherein
"a" represents the measured value of a well tested, "b" represents
the measured value of a cell-free well, and "c" represents the
measured value of a well containing cellss).
[0357] FIGS. 12a, 12b show the results. FIG. 12a shows the result
of the experiment in which goat anti-human IgG (.gamma.)-specific
polyclonal antibodies were not added. While, FIG. 12b shows the
result of the experiment in which goat anti-human IgG
(.gamma.)-specific polyclonal antibodies were added. It was
revealed that the purified human anti-TRAIL-R2 monoclonal antibody
had the cell-death-inducing activity on carcinoma cells compared to
the negative control. Furthermore, it was revealed that the
purified human anti-TRAIL-R2 monoclonal antibodies, 0304 and 0322
induced cell death in carcinoma cells irrespective of the presence
of the cross-linker.
Example 23
Construction of a Vector Expressing the 0304 Antibody of Which
Subclass is IgG1 and the Production of the 0304 Antibody of Which
Subclass is IgG1
[0358] The recombinant 0304 antibody expressing vector constructed
as in Example 14 was converted to N5KG1-Val Lark (IDEC
Pharmaceuticals, the modified vector of N5KG1 (U.S. Pat. No.
6,001,358)) which is an expression vector of IgG1 subclass antibody
by the method described in Example 14. The 0304 antibody (IgG1
type) expressing vector was introduced into a host cell to produce
a cell expressing IgG1 type 0304 antibody. The production and
purification of IgG1 subclass 0304 antibody were performed by the
method described in Example 15.
Example 24
Antigen Binding Activity of the Recombinant 0304 Antibody
[0359] The 0304 antibody of which subclass is IgG4 obtained by the
method described in Examples 14 and 15 and the 0304 antibody of
which subclass is IgG1 obtained by the method described in Example
23 were used to measure the antigen binding activity of the
recombinant 0304 antibody by the method described in Example 18.
For the measurement of the binding activity, 96-well microplate for
ELISA (Maxisorp, Nunc) to which TRAIL-R2-hFc was coated was used.
The results are shown in FIG. 13. FIG. 13 demonstrates that the
amount of the antigen bound to the recombinant 0304 antibody
increases depending on the concentration and the binding activity
is not different in subclasses.
Example 25
Cell-death-inducing Activity on Carcinoma Cells by the Recombinant
0304 Antibody
[0360] Using the 0304 antibody of which subclass is IgG4 obtained
by the method described in Examples 14 and 15 (herein after
referred to as 0304-IgG4) and the 0304 antibody of which subclass
is IgG1 obtained by the method described in Example 23 (herein
after referred to as 0304-IgG1), the cell-death-inducing activity
on Colo205 (ATCC No. CCL-222) cells, which were colon carcinoma
cells, was measured. Colo205 cells cultured in RPMI-1640 medium
containing 10% FCS were prepared at a concentration of
1.0.times.10.sup.5/ml. 100 .mu.l of the suspension was added to
each well of a 96-well flat bottomed plate (Beckton Dickinson).
After culturing at 37.degree. C. under 5.0% carbon dioxide gas for
24 hours, the recombinant antibody 0304 antibody (final
concentration: 10, 30, 100, 300, 1000 ng/ml) was added at 10
.mu.l/well. Furthermore, goat anti-human IgG (.gamma.)-specific
polyclonal antibodies (Sigma) were added (10 .mu.l/well) to each
well at a final concentration of 10 .mu.g/ml. Wells not
supplemented with goat anti-human IgG (.gamma.)-specific polyclonal
antibodies were prepared. After culturing 48 hours at 37.degree. C.
under 5.0% carbon dioxide gas, each well was washed once with PBS
and added with fresh RPMI-1640 medium containing 10% FCS.
Subsequently, an MTS reagent (Cell Titer 968 AQ.sub.UEOUS
Non-Radioactive Cell Proliferation Assay: Promega) was prepared
according to the method described in the instructions, and then 20
.mu.l of the reagent was added to each well. After another 1 hour
of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
absorbance at a wavelength of 490 nm (reference wavelength of 630
nm) was measured using a microplate reader (1420 ARVO multi-label
counter: WALLAC). Using the reducibility of mitochondria as an
indicator, the survival rate of the cells was calculated. The
survival rate of the cells in each well was calculated by the same
formula as disclosed in Example 17.
[0361] FIGS. 14a and 14b show the results. FIG. 14a shows the
result of the experiment in which goat anti-human IgG
(.gamma.)-specific polyclonal antibodies were not added. While,
FIG. 14b shows the result of the experiment in which goat
anti-human IgG (.gamma.)-specific polyclonal antibodies were
added.
[0362] FIG. 14a demonstrates that the recombinant 0304 antibody
alone as a single substance has the cell-death-inducing activity on
Colo205 cells and the activity is not different among subclasses.
Furthermore, FIG. 14b demonstrates that the activity is not
different among subclasses when goat anti-human IgG
(.gamma.)-specific polyclonal antibodies were added. FIG. 14b also
shows that the activity when goat anti-human IgG (.gamma.)-specific
polyclonal antibodies were added is not different with the activity
when goat anti-human IgG (.gamma.)-specific polyclonal antibodies
were not added.
Example 26
Effect of the Recombinant 0304 Antibody on Tumor-bearing Mice
[0363] The effect of the 0304-IgG4 antibody obtained in Examples 14
and 15 and the 0304-IgG1 antibody obtained in Example 23 were
examined using a tumor-bearing mouse model according to the
following method. Colo205, colon carcinoma cells, were
subcutaneously transplanted in the dorsal areas at
5.times.10.sup.6/mouse to 4- to 6-week-old Balb/c nude mice
(purchased from CLEA Japan). Eight or nine days after
transplantation, the sizes of tumors that had adhered were
measured. Five tumor-bearing mice having average tumor sizes of
approximately 100 mm.sup.3 were grouped into a single group. Into
the peritoneal cavities of the tumor-bearing mice, the purified
antibodies were administered at 4, 20 and 100 .mu.g/mouse
(dissolved in 200 .mu.l of PBS), and then the tumor size was
measured. Human anti-DNP antibodies or vehicle (200 .mu.l of PBS
which was used as a medium to dissolve antibody to be administered)
was used as a negative control of the antibody.
[0364] FIGS. 15a and 15b show the results of the above experiments.
In the groups to which 0304-IgG1 antibody had been administered at
4, 20 and 100 .mu.g/mouse three times (administered on days 8, 12
and 16 after transplantation), the reduction of tumor volume was
confirmed in all the mice. With a dose of 100 .mu.g/mouse, the
highest tumor regression effect was observed (FIG. 15a). Changes
with time in tumor volume of the group to which the antibodies were
administered at 100 .mu.g/mouse were as follows.
[0365] On day 4 after the initial administration (corresponding to
day 12 in FIG. 15a), the average tumor volume was 14.447
mm.sup.3;
[0366] On day 7 after the initial administration (corresponding to
day 15 in FIG. 15a), the average tumor volume was 10.693
mm.sup.3:
[0367] On day 10 after the initial administration (corresponding to
day 18 in FIG. 15a), the average tumor volume was 2.976 mm.sup.3:,
and
[0368] On day 13 after the initial administration (corresponding to
day 21 in FIG. 15a), the average tumor volume was 0.556
mm.sup.3.
[0369] The tumor volume on day 4 after the start of administration
was approximately 14.447 mm.sup.3, and a 85% or more tumor
reduction was observed. This reduction was maintained on day 13
after the administration, showing that the 0304-IgG1 antibody
possesses an extremely high anti-tumor effect.
[0370] In the groups to which 0304-IgG4 antibody had been
administered at 4, 20 and 100 .mu.g/mouse three times on alternate
days (administered on days 9, 11 and 13 after transplantation),
anti-tumor activity was observed in all the mice. With a dose of 4
.mu.g/mouse, the tumor volume suppression effect was observed (FIG.
15b). With a dose of 20, 100 .mu.g/mouse, the tumor regression
effect was observed, and with a dose of 20 .mu.g/mouse, the highest
tumor regression effect was observed (FIG. 15b). Changes with time
in tumor volume of the group to which the antibodies were
administered at 20 .mu.g/mouse were as follows.
[0371] On day 2 after the initial administration (corresponding to
day 11 in FIG. 15b), the average tumor volume was 64.814
mm.sup.3;
[0372] On day 4 after the initial administration (corresponding to
day 13 in FIG. 15b), the average tumor volume was 38.590
mm.sup.3:
[0373] On day 6 after the initial administration (corresponding to
day 15 in FIG. 15b), the average tumor volume was 27.923 mm.sup.3:,
and On day 9 after the initial administration (corresponding to day
18 in FIG. 15b), the average tumor volume was 26.316 mm.sup.3, and
On day 12 after the initial administration (corresponding to day 21
in FIG. 15b), the average tumor volume was 40.076 mm.sup.3.
[0374] The tumor volume on day 2 after the start of administration
was approximately 64.814 mm.sup.3, and a 35% or more tumor
reduction was observed. This reduction was maintained on day 12
after the administration, showing that the 0304-IgG4 antibody
possesses a high anti-tumor effect like 0304-IgG1 antibody. The
0304-IgG1 antibody shows higher anti-tumor effect. From this fact,
it is speculated that CDC and/or ADCC which are assumed to be
possessed by IgG1 subclass act on 0304-IgG1.
Example 27
Fractionation of Monomer Antibody
[0375] 0304 antibody and 0322 antibody obtained in Example 21 as
well as H-48-2 antibody (deposit No.: FERM BP-7599) were
fractionated using HPLC (L-7120, Hitachi). Each antibody was added
at flow rate of 0.25 ml/min. to Superdex 200HR column (Amersham
Pharmacia Biotech) equilibrated with PBS, and eluted at flow rate
of 0.25 ml/min. with PBS. When eluted, a fraction collector
(FC203B, GILSON) was used to collect eluted fractions at 0.5
ml/fraction. The collected fractions were filtered for
sterilization with a membrane filter (ULTRAFREE-MC STERILE
(Millipore) having a pore size of 0.22 .mu.m. Then, absorbance at
280 nm was measured and the antibody concentration of each fraction
was calculated using 1.4 OD=1 mg/ml. FIGS. 16a, 16b and 16c show
the result of the fractionation of each antibody.
Example 28
Cell-death-inducing Activity on Carcinoma Cells by the Fractionated
Antibody
[0376] Using the fractionated antibody obtained in Example 27, the
cell-death-inducing activity on Colo205 (ATCC No. CCL-222) cells,
which were colon carcinoma cells, was measured. Colo205 cells
cultured in RPMI-1640 media containing 10% FCS were prepared at a
concentration of 1.0.times.10.sup.5/ml. 100 .mu.l of the suspension
was added to each well of a 96-well flat bottomed plate (Beckton
Dickinson). After culturing at 37.degree. C. under 5.0% carbon
dioxide gas for 24 hours, the fractionated antibody (final
concentration: 1000 ng/ml) was added at 10 .mu.l/well. Furthermore,
goat anti-human IgG (.gamma.)-specific polyclonal antibodies
(Sigma) were added (10.mu.l/well) at a final concentration of 10
.mu.g/ml to each well. Wells not supplemented with goat anti-human
IgG (.gamma.)-specific polyclonal antibodies were prepared. In the
measurement of H-48-2 antibody, for the fractions of which protein
concentration was low (fraction Nos. 20-25, 32 and 33) as a result
of the fractionation, the eluates which were sterilized by
filtration were directly added into wells (10 .mu.l/well). After 48
hours of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
each well was washed once with PBS and added with fresh RPMI-1640
medium containing 10% FCS. Subsequently, an MTS reagent (Cell Titer
96.RTM. AQ.sub.UEOUS Non-Radioactive Cell Proliferation Assay:
Promega) was prepared according to the method described in the
instructions. 20 .mu.l of the reagent was added to each well. After
2 hours of culturing at 37.degree. C. under 5.0% carbon dioxide
gas, absorbance at a wavelength of 490 nm (with a reference
wavelength of 630 nm) was measured using a microplate reader (1420
ARVO multi-label counter: WALLAC). Using the reducibility of the
mitochondria as an indicator, the survival rate of the cells was
calculated. The survival rate of the cells in each well was
calculated using a formula similar to that of Example 17.
[0377] FIGS. 17a to 17f show the results. FIGS. 17a, 17c and 17e
show the results of an experiment wherein no goat anti-human
IgG(.gamma.)-specific polyclonal antibody was added, and FIGS. 17b,
17d and 17f show the result of an experiment wherein goat
anti-human IgG(.gamma.)-specific polyclonal antibodies were
added.
[0378] As shown in FIG. 17a, for 0304 antibody, it was revealed
that not only the fractions containing dimer or multimer antibody
(hereinafter referred to as antibody polymer) (in FIG. 17a,
fraction Nos. 23-25 of X axis) but also the fractions containing
monomer (in FIG. 17a, fraction Nos. 26-32 of X axis) had the
cell-death-inducing activity on Colo205 cells. Furthermore, as
shown in FIG. 17b, the fractions which have the activity when added
with anti-human IgG(.gamma.)-specific polyclonal antibody were the
same with the fractions which have the activity when anti-human
IgG(.gamma.)-specific polyclonal antibody was not added.
[0379] As shown in FIG. 17c, for 0322 antibody, it was revealed
that not only the fractions containing antibody polymer (in FIG.
17c, fraction Nos. 22-25 of X axis) but also the fractions
containing monomer (in FIG. 17c, fraction Nos. 26-32 of X axis) had
the cell-death-inducing activity on Colo205 cells. Furthermore, as
shown in FIG. 17d, the fractions which have the activity when added
with anti-human IgG(.gamma.)-specific polyclonal antibody were the
same with the fractions which have the activity when anti-human
IgG(.gamma.)-specific polyclonal antibody was not added.
[0380] On the contrary, as shown in FIG. 17e, for H-48-2 antibody,
it was revealed that the fractions containing antibody polymer (in
FIG. 17e, fraction Nos. 21-25 of X axis) had the
cell-death-inducing activity on Colo205 cells but the fractions
containing monomer (in FIG. 17c, fraction Nos. 26-31 of X axis) did
not have the cell-death-inducing activity on Colo205 cells. As
shown in FIG. 17f, when anti-human IgG(.gamma.)-specific polyclonal
antibody was added, both of the fractions containing polymer
antibody and monomer had the cell-death-inducing activity on
Colo205 cells.
[0381] Therefore, it was revealed that the monomer of 0304 antibody
and 0322 antibody had the cell-death-inducing activity on Colo205
cells, but the monomer of H-48-2 antibody did not have the
cell-death-inducing activity on Colo205 cells and H-48-2 antibody
exhibited the cell-death-inducing activity when it was
cross-linked.
[0382] The same experiments were performed for the fractions which
were obtained by the method described in Example 27 from 0304-IgG1
antibody obtained by Example 23 and the same results as the above
0304 antibody were obtained.
[0383] Consequently, it was found that 0304 antibody and 0322
antibody had the cell-death-inducing activity as a monomer. It was
also found that the activity possessed by a monomer of 0304
antibody did not depend on the antibody subclass and the cells
producing the antibody.
Example 29
The Requirement of a Cross-linker for the Fractionated
Antibody-mediated Cell Death
[0384] , Regarding the requirement of cross-linking for the
cell-death-inducing activities of the monomer fractions of the
human anti-TRAIL-R2 antibodies, 0304-IgG1 and H-48-2 obtained by
the method described in Example 27, it was examined whether or not
the activity was neutralized in the presence of a competition
antibody (control IgG: anti HSA human antibody).
[0385] Colo205 cells cultured in RPMI-1640 medium containing 10%
FCS were collected by Trypsin-EDTA treatment and prepared at a
concentration of 5.times.10.sup.4/ml. Then, the cells were added
into 96-well flat-bottomed plate (Beckton Dickinson) at 100
.mu.l/well. After 24 hours of culturing at 37.degree. C. under 5.0%
carbon dioxide gas, the monomer fraction of human anti-TRAIL-R2
monoclonal antibody was added at the final concentration of 1
.mu.g/ml to each well (10 .mu.l/well). After 0.5-1 hours of
culturing at 37.degree. C. under 5.0% carbon dioxide gas, excessive
amount of a competition antibody (control IgG1) was added at the
final concentration of 0, 3, 10, 30 and 100 .mu.g/ml to each well,
and goat anti-human IgG (.gamma.)-specific polyclonal antibody or
human peripheral blood cells (hereinafter, referred to as hPBMC)
which are thought to be endogenous cross-linker were subsequently
added as a cross-linker. The final concentration of goat anti-human
IgG (.gamma.)-specific polyclonal antibody was 10 .mu.g/ml, and the
final concentration of hPBMC was 2.5.times.10.sup.5/well, which was
50 times the amount of carcinoma cells. As a comparative control,
the wells to which the antibody was added alone were prepared.
After the addition of hPBMC, the plate was gently shaken such that
the carcinoma cells were not detached, and cultured at 37.degree.
C. under 5.0% carbon dioxide gas. After culturing for 2 days, each
well was washed once with PBS and added with fresh RPMI-1640.
medium containing 10% FCS. Subsequently, 20 IAl of MTS
reagent.(Cell Titer 96.RTM. AQ.sub.UEOUS Non-Radioactive Cell
Proliferation Assay: Promega) was added to each well. After
additional 2.5-3.5 hours of culturing at 37.degree. C. under 5.0%
carbon dioxide gas, absorbance at a wavelength of 490 nm (reference
wavelength of 630 nm) was measured using a microplate reader
(SPECTRA MAX 250: Molecular Devices). Using the reducibility of
mitochondria as an indicator, the survival rate of the cells was
calculated. The survival rate of the cells in each well was
calculated by the following formula: Survival rate
(%)=100.times.(a-b)/(c-b) (wherein "a" represents the measured
value of a well tested, "b" represents the measured value of a
cell-free well, and "c" represents the measured value of a well
containing Colo205 cells, in this example the measured value of a
well in which carcinoma cells were cultured with medium only).
FIGS. 18a and 18b show the results. In the figures, aHSA means the
results treated with an anti-HSA human IgG1 antibody used as the
competition antibody and Ab alone means the results treated only
with the monomer fraction of the antibody.
[0386] FIG. 18a shows the cell-death inducing activity of each
anti-TRAIL-R2 antibody when Goat anti-human IgG (.gamma.) specific
polyclonal antibody was used as a cross-linker in the presence of
each concentration of the competition antibody. FIG. 18b shows the
cell-death inducing activity of each anti-TRAIL-R2 antibody when
human PBMC was used as a cross-linker in the presence of each
concentration of the competition antibody As shown in FIG. 18a, the
monomer fraction of H-48-2-IgG1 of which monomer fraction alone
have no activity (hereinafter, referred to as monomer inactive
antibody) did not have the cell-death-inducing activity as a single
agent also in this study. It had the cell-death-inducing activity
when goat anti-human IgG (.gamma.)-specific polyclonal antibody was
co-existed as a cross-linker. Furthermore, the activity in the
presence of goat anti-human IgG (.gamma.)-specific polyclonal
antibody as a cross-linker was neutralized by the competition
antibody in a concentration-dependent manner. This indicates that
the activity of H-48-2 is affected by the cross-linker. On the
contrary, the monomer fraction of 0304-IgG1 had the activity when
used alone and also had the activity in the presence of the
cross-linker. Furthermore, the activity in the presence of goat
anti-human IgG (.gamma.)-specific polyclonal antibody as a
cross-linker was not neutralized at all by the competition
antibody. This indicates that the activity of 0304 is not affected
by the cross-linker.
[0387] The same findings were also observed when human PBMC was
uses as a cross-linker.
[0388] As FIG. 18b shows, the monomer fraction of H-48-2 antibody
which is monomer inactive antibody had no cell-death-inducing
activity when used alone but had the activity when used in the
presence of hPBMC as a cross-linker. The activity in the presence
of hPBMC as a cross-linker was neutralized by excess amount of the
competition antibody. This suggests that the activity of H-48-2 is
affected by an endogenous cross-linker. Furthermore, since the
neutralization by the competition antibody was caused by masking Fc
receptor (hereinafter, referred to as FcR) on effecter cells in
hPBMC, it is thought that FcR is involved in the cross-linking. On
the contrary, 0304. which is the antibody of which monomer fraction
has the activity (hereinafter reffered to as monomer active
antibody) has the activity as a monomer fraction alone
independently of hPBMC. Furthermore, since the activity in the
presence of hPBMC was not neutralized at all by excessive amount of
the competition antibody, the activity of 0304 was revealed not to
be affected by an endogenous cross-linker.
[0389] Consequently, it was shown that the activity of monomer
active antibody, unlike monomer inactive antibody, is not affected
by hPBMC which is thought to be an endogenous cross-linker as well
as goat anti-human IgG (.gamma.)-specific polyclonal antibody which
is thought to be an artificial cross-linker, and the activity is
expected independently of a cross-linker.
Example 30
The Requirement of the Cross-linking by Fc Receptors for the
Cell-death-inducing Activity
[0390] Regarding the cell-death-inducing activity of the monomer
fraction of the human anti-TRAIL-R2 antibody, 0304-IgG1 and H-48-2,
which were obtained by the method described in Example 27, the
requirement of hPBMC (including Fc receptor positive cells) which
is thought to be an endogenous cross-linker and the contribution of
cross-linking by FcR were examined. Colo205 cells which had been
cultured in RPMI-1640 medium containing 10% FCS were collected by
Trypsin-EDTA treatment and prepared at the concentration of
5.times.10.sup.4/ml. The cells were added to each well of 96-well
flat-bottomed plate (Beckton Dickinson) at 100 .mu.l/well. After 24
hours of culturing at 37.degree. C. under 5.0% carbon dioxide gas,
a monomer fraction of the human anti-TRAIL-R2 monoclonal antibody
and the unfractionated anti-DNP human IgG1 antibody as a negative
control were added at the final concentration of 1 .mu.g/ml to each
well (10 .mu.l/well). After 0.5-1 hours of culturing at 37.degree.
C. under 5.0% carbon dioxide gas, the suspension of human PBMC was
added at 10 .mu.l/well to adjust E/T ratio (E: effector cell, T:
target cell, E and T show respectively, human PBMC and a carcinoma
cell in this example) the value indicated in figures. As a
comparative control, the wells to which the antibody was added
alone and wells added with goat anti-human IgG (.gamma.)-specific
polyclonal antibody as a cross-linker were prepared. Furthermore,
to investigate whether or not the cross-linking by human PBMC is
involved by Fc receptor, some hPBMCs was treated with anti-CD16
antibody (Pharmingen)/anti-CD32 antibody (Selotec)/ anti-CD64
antibody (Caltag Laboratories) mixture (hereinafter, reffered to as
FcR blocker) at each concentration of 10 .mu.g/ml per
1.times.10.sup.7 cells for more than 30 minutes. Then, the cell
suspension was added without being washed. Alternatively, excessive
amount (100 times the amount of anti-TRAIL-R2 antibody) of a
competition antibody (control IgG1: anti-HSA human antibody) was
added for the same purpose. After the addition of the human PBMC,
the plate was gently shaken such that the carcinoma cells were not
detached, and cultured at 37.degree. C. under 5.0% carbon dioxide
gas. After culturing for 2 days, each well was washed mildly with
PBS and added with fresh RPMI-1640 medium containing 10% FCS.
Consequently, 20 .mu.l of MTS reagent (Cell Titer 96.RTM.
AQ.sub.UEOUS Non-Radioactive Cell Proliferation Assay: Promega) was
added to each well. After additional 2-3 hours of culturing at
37.degree. C. under 5.0% carbon dioxide gas, absorbance at a
wavelength of 490 nm (reference wavelength of 630 nm) was measured
using a microplate reader (SPECTRA MAX 250: Molecular Devices).
Using the reducibility of mitochondria as an indicator, the
survival rate of the cells was calculated using the formula of
Example 29.
[0391] FIGS. 19a and 19b show the results. In the figures, Ab alone
means the results treated only with the monomer fraction of the
antibody and anti hg pAb means the results in case where goat
anti-human IgG specific polyclonal antibody was added as a
cross-linker together with the monomer fraction of the antibody.
FIG. 19a shows the survival rate of Colo205 cells which was
cultured for 2 days with 1 .mu.g/ml of each antibody and with hPBMC
of which E/T ratio was 12.5:1, 25:1 or 50:1 in the presence of
excessive amount of the competition antibody (anti HSA human IgG1)
(in the figure, represented by Competition (+)) or the absence of
the competition antibody (in the figure, represented by Competition
(-)). FIG. 19b shows the survival rate of Colo205 cells which was
cultured for 2 days with 1 .mu.g/ml of each antibody and with the
human PBMC of which E/T ratio was 10:1, 20:1 or 40:1, and which was
treated with FcR blocker (FcR-block (+)) or not (FcR block
(-)).
[0392] As shown in FIGS. 19a, the monomer fraction of H-48-2 which
was monomer inactive antibody did not have the cell-death-inducing
activity alone also in this study. It had the cell-death-inducing
activity when goat anti-human IgG (.gamma.)-specific polyclonal
antibody or hPBMC was co-existed as a cross-linker. This shows that
the activity of H-48-2 is affected by a cross-linker. Furthermore,
since the activity in the presence of hPBMC as a cross-linker was
neutralized in the presence of excessive amount of the competition
antibody, it was shown that FcR is involved in cross-linking. On
the contrary, 0304-IgG1 which was monomer active antibody had the
activity when used as an antibody alone independently of hPBMC and
the activity was not neutralized at all even in the presence of
super excessive competition antibody. From the results, it is
confirmed that the activity of 0304 is not affected by a
cross-linker.
[0393] The same findings were also observed in the results shown in
FIG. 19b. That is, the monomer fraction of H-48-2 which is monomer
inactive antibody had no cell-death-inducing activity used as a
single substance at a concentration of 1 .mu.g/ml, but had the
activity when used in the presence of hPBMC as a cross-linker. This
shows that the activity of H-48-2 is affected by a cross-linker.
Furthermore, the activity when hPBMC was co-existed as a
cross-linker was neutralized by previously treating hPBMC with FcR
blocker. This shows that FcR is involved in cross-linking. On the
contrary, 0304-IgG1 had the activity when used alone independently
of hPBMC and the activity was not neutralized at all by the
pretreatment of hPBMC with FcR blocker. This clearly supports the
findings that the activity of 0304 does not depend on a
cross-linker.
[0394] As shown in Example 28, 0304 and 0322 are the antibodies
characterized in that a monomer antibody alone has the
cell-death-inducing activity. Furthermore, as shown in Example 26,
it was confirmed that 0304 had significant anti-tumor effect on
tumor-bearing mice both as IgG1 subclass and IgG4 subclass. Hence,
0304 which has the cell-death-inducing activity and anti-tumor
activity as a monomer independently of exogenous factors is
expected to be able to exert the anti-tumor activity independent on
the physiological conditions (e.g., types or the number of
immunocompetent cells) of a patient to which a prophylactic or
therapeutic agent against disease caused by TRAIL-R2-expressing
cells is to be administered, particularly when a therapeutic agent
against malignant tumor is to be administered.
Example 31
The Activity of a Monomer Active Antibody for Inducing Aggregation
of TRAIL-R2 Molecules on the Surface of Cells
[0395] To investigate the affect of the treatment of an antibody on
behavior of a receptor molecule which exists on the surface of
cells, 0304 which is a monomer active antibody. H-48-2 which is a
monomer inactive antibody and a chemical cross-linker (DTSSP,
Pierce) were used to perform the following studies.
6.times.10.sup.6 cells of Colo205 cells, which were human colon
carcinoma cells, were suspended in 0.3 ml of PBS, 7.2 .mu.g of each
antibody in the monomer fraction of human anti-TRAIL-R2 antibodies,
0304-IgG1 and H-48-2, obtained by the method described in Example
27 was added to the suspension and the cells were incubated at
37.degree. C. for 10minutes. Then, the cell suspension was
centrifuged to remove the supernatant containing unbound antibodies
and the suspension was re-suspended in 0.5 ml of PBS to wash and
remove the unbound antibodies. The thus obtained cell suspension
was suspended in 0.3 ml of PBS and a chemical cross-linker solution
(200 mM DTSSP) was added to the suspension at a final concentration
of 2 mM. After standing the suspension on ice for 2 hours, 1M
Tris-HCl pH7.5 was added at a final concentration of 50 mM to
inactivate the excessive chemical cross-linker and the suspension
was stood on ice for 15 minutes. Then, the cells were collected by
centrifugation, the supernatant was removed, the cell lysing
solution (PBS containing 0.1% Triton X-100), 9 times the volume of
cell pellet, was added, cells were completely suspended and the
suspension was stood on ice for 30 minutes. After the increase of
transparency of the cell suspension was observed, the suspension
was centrifuged for 30 minutes to remove insoluble fraction. The
obtained soluble fraction was referred to as cell lysate solution.
100 .mu.l of the lysate solution was subjected to gel filtration
analysis and the distribution of the molecular weight of
antibody-antigen complex which were cross-linked was
investigated.
[0396] The gel filtration conditions were as follows.
[0397] HPLC System: A pump and detector are Hitachi L-6000
series.
[0398] Gel filtration column: Superose 6
(Amersham.multidot.Bioscience, 1 cm of diameter.times.30 cm of
length
[0399] Equilibrated buffer: 20 mM phosphate buffer pH7.0, 200 mM
NaCl
[0400] Flow rate: 0.5 ml/min.
[0401] Detection: UV280 nm
[0402] After the eluted protein fraction was collected by gel
filtration, 50 .mu.l of each fraction was subjected to human IgG
ELISA to detect an antibody-antigen complex and analyze the
distribution of a molecular weight. The human IgG ELISA was
performed by the method described in Example 19 by using rabbit
anti-human IgG polyclonal antibody (DAKO) as an capture antibody
and HRP-labeled rabbit anti-human IgG polyclonal F(ab').sub.2
antibody (DAKO) as a detection antibody. The result was shown in
FIG. 20.
[0403] As shown in FIG. 20, in the incubation of the monomer
fraction of H-48-2 which is a monomer inactive antibody with
Colo205 cells, antibody-antigen complexes were hardly detected in
fractions Nos. 3 and 4 (more than 4000 kDa of molecular weight)
which are corresponding to void of gel filtration column. On the
contrary, in the incubation of 0304 which is a monomer active
antibody, a peak was clearly observed at that position. It was also
shown that antibody-antigen complex of 0304 is clearly superior to
that of H-48-2 in quantity in the fractions of higher molecular
weight, fractions Nos. 5 to 11. These results show that 0304 which
is a monomer active antibody can readily form a larger
antibody-antigen complex on the surface of cells than H-48-2 which
in a monomer inactive antibody and therefore 0304 has a tendency to
induce a large aggregate of TRAIL-R2 which is an antigen. It has
been known that the aggregation of TRAIL-R2 on the surface of cells
induces cell death. Therefore, the possibility that the induction
of cell death by a monomer active antibody is strongly involved in
the aggregating activity of the receptor molecule was suggested.
The fraction No. 19 is corresponding to the eluate peak of the
antibody alone. The elution of the antibody alone was observed for
both of 0304 and H-48-2. From the fact, it is assumed that the
molecules which were not cross-linked were dissociated from the
antigens during the preparation of cell lysate solution and the
antibody alone was collected as a result of the fractionation by
gel filtration since the frequency of the intermolecular binding by
cross-linker is 5 to 20%.
[0404] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
[0405] Industrial Applicability
[0406] According to the present invention, there is provided a
molecule with extremely high safety, which is useful as a
prophylactic or therapeutic agent against disease, in particular
malignant tumors, caused by TRAIL-R1 and R2-expressing cells, and
which can avoid damage to the liver.
[0407] There is also provided a molecule, which is useful as a
prophylactic or therapeutic agent against disease caused by
TRAIL-R1 and/or R2-expressing cells, had the activity to induce
apoptosis by adding or administering a monomer antibody
independently exogenous factors and is expected to have a strong
high anti-tumor effect.
[0408] Sequence Listing Free Text
[0409] SEQ ID NO: 1: synthetic DNA
[0410] SEQ ID NO: 2: synthetic DNA
[0411] SEQ ID NO: 3: synthetic DNA
[0412] SEQ ID NO: 4: synthetic DNA
[0413] SEQ ID NO: 5: synthetic DNA
[0414] SEQ ID NO: 6: synthetic DNA
[0415] SEQ ID NO: 7: synthetic DNA
[0416] SEQ ID NO: 8: synthetic DNA
[0417] SEQ ID NO: 9: synthetic DNA
[0418] SEQ ID NO: 10: synthetic DNA
[0419] SEQ ID NO: 11: synthetic DNA
[0420] SEQ ID NO: 12: synthetic DNA
[0421] SEQ ID NO: 13: synthetic DNA
[0422] SEQ ID NO: 14: synthetic DNA
[0423] SEQ ID NO: 15: synthetic DNA
[0424] SEQ ID NO: 36: synthetic DNA
[0425] SEQ ID NO: 37: synthetic DNA
[0426] SEQ ID NO: 38: synthetic DNA
[0427] SEQ ID NO: 39: synthetic DNA
[0428] SEQ ID NO: 40: synthetic DNA
[0429] SEQ ID NO: 41: synthetic DNA
[0430] SEQ ID NO: 42: synthetic DNA
[0431] SEQ ID NO: 43: synthetic DNA
[0432] SEQ ID NO: 44: synthetic DNA
[0433] SEQ ID NO: 45: synthetic DNA
Sequence CWU 1
1
45 1 30 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 1 cacgaattca ccatggcgcc accaccagct 30 2 48
DNA Artificial Sequence Description of Artificial SequenceSynthetic
DNA 2 tttctcgagg cggccgctta tcactccaag gacacggcag agcctgtg 48 3 33
DNA Artificial Sequence Description of Artificial SequenceSynthetic
DNA 3 cacgaattcg ccaccatgga acaacgggga cag 33 4 48 DNA Artificial
Sequence Description of Artificial SequenceSynthetic DNA 4
tttctcgagg cggccgctca ttaggacatg gcagagtctg cattacct 48 5 37 DNA
Artificial Sequence Description of Artificial SequenceSynthetic DNA
5 ttctacgagc ggcttatcac agcctcctcc tctgaga 37 6 40 DNA Artificial
Sequence Description of Artificial SequenceSynthetic DNA 6
ttctacgagc ggccgcttat cacaagtctg caaagtcatc 40 7 27 DNA Artificial
Sequence Description of Artificial SequenceSynthetic DNA 7
ggtccgggag atcatgaggg tgtcctt 27 8 26 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 8 gtgcacgccg
ctggtcaggg cgcctg 26 9 23 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 9 ggtgccaggg ggaagaccga tgg 23 10
34 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 10 atatagatct ctcagttagg acccagaggg aacc 34
11 31 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 11 gatgggccct tggtgctagc tgaggagacg g 31 12
26 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 12 gttgaagctc tttgtgacgg gcgagc 26 13 26 DNA
Artificial Sequence Description of Artificial SequenceSynthetic DNA
13 tggcgggaag atgaagacag atggtg 26 14 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 14 atatgtcgac
tacggggggg ctttctgaga gtc 33 15 32 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 15 aagacagatg
gtgcagccac cgtacgtttg at 32 16 467 DNA Homo sapiens 16 gtcgactacg
ggggggcttt ctgagagtca tggatctcat gtgcaagaaa atgaagcacc 60
tgtggttctt cctcctgctg gtggcggctc ccagatgggt cctgtcccag ctgcagctgc
120 aggagtcggg cccaggactg gtgaagcctt cggagaccct gtccctcacc
tgcactgtct 180 ctggtggctc catcatcagt aaaagttcct actggggctg
gatccgccag cccccaggga 240 aggggctgga gtggattggg agtatctatt
atagtgggag taccttctac aacccgtccc 300 tcaagagtcg agtcaccata
tccgtagaca cgtccaagaa ccagttctcc ctgaagctga 360 gctctgtgac
cgccgcagac acggctgtgt attactgtgc gagactgaca gtggctgagt 420
ttgactactg gggccaggga accctggtca ccgtctcctc agctagc 467 17 146 PRT
Homo sapiens 17 Met Asp Leu Met Cys Lys Lys Met Lys His Leu Trp Phe
Phe Leu Leu 1 5 10 15 Leu Val Ala Ala Pro Arg Trp Val Leu Ser Gln
Leu Gln Leu Gln Glu 20 25 30 Ser Gly Pro Gly Leu Val Lys Pro Ser
Glu Thr Leu Ser Leu Thr Cys 35 40 45 Thr Val Ser Gly Gly Ser Ile
Ile Ser Lys Ser Ser Tyr Trp Gly Trp 50 55 60 Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr 65 70 75 80 Tyr Ser Gly
Ser Thr Phe Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr 85 90 95 Ile
Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser 100 105
110 Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Thr Val
115 120 125 Ala Glu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 130 135 140 Ala Ser 145 18 421 DNA Homo sapiens 18
tcacagatct ctcagttagg acccagaggg aaccatggaa gccccagctc agcttctctt
60 cctcctgcta ctctggctcc cagataccac cggagaaatt gtgttgacac
agtctccagc 120 caccctgtct ttgtctccag gggaaagagc caccctctcc
tgcagggcca gtcagagtgt 180 tagcagcttc ttagcctggt accaacagaa
acctggccag gctcccaggc tcctcatcta 240 tgatgcatcc aacagggcca
ctggcatccc agccaggttc agtggcagtg ggtctgggac 300 agacttcact
ctcaccatca gcagcctaga gcctgaagat tttgcagttt attactgtca 360
gcagcgtagc aactggcctc tcactttcgg ccctgggacc aaagtggata tcaaacgtac
420 g 421 19 129 PRT Homo sapiens 19 Met Glu Ala Pro Ala Gln Leu
Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val
Ser Ser Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55
60 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser 100 105 110 Asn Trp Pro Leu Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Lys Arg 115 120 125 Thr 20 467 DNA Homo sapiens 20
gtcgactacg ggggggcttt ctgagagtca tggatctcat gtgcaagaaa atgaagcacc
60 tgtggttctt cctcctgctg gtggcggctc ccagatgggt cctgtcccag
ttgcagctgc 120 aggagtcggg cccaggactg gtgaagccct cggagaccct
gtccctcacc tgcactgtct 180 ctggtggctc catcagcagt aggagtaact
actggggctg gatccgccag cccccaggga 240 aggggctgga gtggattggg
aatgtctatt atagagggag cacctactac aattcgtccc 300 tcaagagtcg
agtcaccata tccgtagaca cgtccaagaa ccagttctcc ctgaagctga 360
gctctgtgac cgtcgcagac acggctgtgt attactgtgc gagactgtca gtggctgagt
420 ttgactactg gggccaggga atcctggtca ccgtctcctc agctagc 467 21 146
PRT Homo sapiens 21 Met Asp Leu Met Cys Lys Lys Met Lys His Leu Trp
Phe Phe Leu Leu 1 5 10 15 Leu Val Ala Ala Pro Arg Trp Val Leu Ser
Gln Leu Gln Leu Gln Glu 20 25 30 Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu Thr Leu Ser Leu Thr Cys 35 40 45 Thr Val Ser Gly Gly Ser
Ile Ser Ser Arg Ser Asn Tyr Trp Gly Trp 50 55 60 Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile Gly Asn Val Tyr 65 70 75 80 Tyr Arg
Gly Ser Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr 85 90 95
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser 100
105 110 Val Thr Val Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ser
Val 115 120 125 Ala Glu Phe Asp Tyr Trp Gly Gln Gly Ile Leu Val Thr
Val Ser Ser 130 135 140 Ala Ser 145 22 417 DNA Homo sapiens 22
agatctctca gttaggaccc agagggaacc atggaagccc cagctcagct tctcttcctc
60 ctgctactct ggctcccaga taccaccgga gaaattgtgt tgacacagtc
tccagccacc 120 ctgtctttgt ctccagggga aagagccacc ctctcttgta
gggccagtca gagtgttagc 180 agcttcttag cctggtacca acagaaacct
ggccaggctc ccaggctcct catctatgat 240 gcatccaaca gggccactgg
cagcccagcc aggttcagtg gcagtgggtc tgggacagac 300 ttcactctca
ccatcagcag cctagagcct gaagattttg cagtttatta ctgtcagcag 360
cgtagcgact ggcctctcac tttcggccct gggaccaaag tggatatcaa acgtacg 417
23 129 PRT Homo sapiens 23 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu
Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser Ser Phe
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu
Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ser Pro Ala 65 70 75 80
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85
90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser 100 105 110 Asp Trp Pro Leu Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 115 120 125 Thr 24 490 DNA Homo sapiens 24 tcgactacgg
gggggctttc tgagagtcat ggatctcatg tgcaagaaaa tgaagcacct 60
gtggttcttc ctcctgctgg tggcggctcc cagatgggtc ctgtcccagc tgcagctgca
120 ggagtcgggc ccaggactgg tgaagccttc ggagaccctg tccctcacct
gcactgtctc 180 tggtggctcc atcagcagta gtagttacta ctggggctgg
gtccgccagc ccccagggaa 240 ggggctggag tggattggga gtatccatta
tagtgggagt actttctaca acccgtccct 300 caagagtcga gtcaccattt
ccgtagacac gtccaagaac cagttctccc tgaagctgag 360 ctctgtgacc
gccgcagaca cgactgtgta ttactgtgcg agacaggggt ctactgtggt 420
tcggggagtt tactactacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc
480 ctcagctagc 490 25 154 PRT Homo sapiens 25 Met Asp Leu Met Cys
Lys Lys Met Lys His Leu Trp Phe Phe Leu Leu 1 5 10 15 Leu Val Ala
Ala Pro Arg Trp Val Leu Ser Gln Leu Gln Leu Gln Glu 20 25 30 Ser
Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 35 40
45 Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly Trp
50 55 60 Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser
Ile His 65 70 75 80 Tyr Ser Gly Ser Thr Phe Tyr Asn Pro Ser Leu Lys
Ser Arg Val Thr 85 90 95 Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu Lys Leu Ser Ser 100 105 110 Val Thr Ala Ala Asp Thr Thr Val
Tyr Tyr Cys Ala Arg Gln Gly Ser 115 120 125 Thr Val Val Arg Gly Val
Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 130 135 140 Gly Thr Thr Val
Thr Val Ser Ser Ala Ser 145 150 26 423 DNA Homo sapiens 26
agatctctca gttaggaccc agagggaacc atggaaaccc cagcgcagct tctcttcctc
60 ctgctactct ggctcccaga taccaccgga gaaattgtgt tgacgcagtc
tccaggcacc 120 ctgtctttgt ctccagggga aagagccacc ctctcctgca
gggccagtca gagtgttagc 180 agcagctact tagcctggta ccagcagaaa
cctggccagg ctcccaggct cctcatctat 240 ggtgcatcca gcagggccac
tggcatccca gacaggttca gtggcagtgg gtctgggaca 300 gacttcactc
tcaccatcag cagactggag cctgaagatt ttgcagtgta ttactgtcag 360
cagtatggta gctcacctct gtacactttt ggccagggga ccaagctgga gatcaaacgt
420 acg 423 27 131 PRT Homo sapiens 27 Met Glu Thr Pro Ala Gln Leu
Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45 Val
Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55
60 Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro
65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr 100 105 110 Gly Ser Ser Pro Leu Tyr Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr 130 28 489 DNA Homo
sapiens 28 ctcaacaacc acatctgtcc tctagagaaa accctgtgag cacagctcct
caccatggac 60 tggacctgga ggatcctctt cttggtggca gcagctacaa
gtgcccactc ccaggtgcag 120 ctggtgcagt ctggggctga gatgaagaag
cctggggcct cagtcaaggt ctcctgcaag 180 acttctggat acaccttcac
caattataag atcaactggg tgcgacaggc ccctggacaa 240 ggacttgagt
ggatgggatg gatgaaccct gacactgata gcacaggcta tccacagaag 300
ttccagggca gagtcaccat gaccaggaac acctccataa gcacagccta catggagctg
360 agcagcctga gatctgagga cacggccgtg tattactgtg cgagatccta
tggttcgggg 420 agttattata gagactatta ctacggtatg gacgtctggg
gccaagggac cacggtcacc 480 gtctcctca 489 29 145 PRT Homo sapiens 29
Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Ser 1 5
10 15 Ala His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Met Lys
Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Tyr Thr Phe 35 40 45 Thr Asn Tyr Lys Ile Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp Met Asn Pro Asp
Thr Asp Ser Thr Gly Tyr Pro 65 70 75 80 Gln Lys Phe Gln Gly Arg Val
Thr Met Thr Arg Asn Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys
Ala Arg Ser Tyr Gly Ser Gly Ser Tyr Tyr Arg Asp Tyr 115 120 125 Tyr
Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135
140 Ser 145 30 417 DNA Homo sapiens 30 gaggaactgc tcagttagga
cccagaggga accatggaag ccccagctca gcttctcttc 60 ctcctgctac
tctggctccc agataccacc ggagaaattg tgttgacaca gtctccagcc 120
accctgtctt tgtctccagg ggaaagagcc accctctcct gcagggccag tcagagtgtt
180 agcagctact tagcctggta ccaacagaaa cctggccagg ctcccaggct
cctcatctat 240 gatgcatcca acagggccac tggcatccca gccaggttca
gtggcagtgg gtctgggaca 300 gacttcactc tcaccatcag cagcctagag
cctgaagatt ttgcagttta ttactgtcag 360 cagcgtagca actggccgct
cactttcggc ggagggacca aggtggagat caaacga 417 31 128 PRT Homo
sapiens 31 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp
Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser 35 40 45 Val Ser Ser Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60 Arg Leu Leu Ile Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95 Ser Leu
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 100 105 110
Asn Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 115
120 125 32 497 DNA Homo sapiens 32 gagctctgag agaggagccc agccctggga
ttttcaggtg ttttcatttg gtgatcagga 60 ctgaacagag agaactcacc
atggagtttg ggctgagctg gctttttctt gtggctattt 120 taaaaggtgt
ccagtgtgag gtacagctgt tggagtctgg gggaggcttg gtacagcctg 180
ggaggtccct gagactctcc tgtgcagcct ctggattcac ctttagcagc tatgccatga
240 gctgggtccg ccaggctcca gggaaggggc tggagtgggt ctcagctatt
agtggtagtg 300 gtggtagcag atactacgca gactccgtga agggccggtt
caccatctcc agagacaatt 360 ccaagaacac gctgtatctg caaatgaaca
gcctgagagc cgaggacacg gccgtatatt 420 actgtgcgaa agagagcagt
ggctggttcg gggcctttga ctactggggc cagggaaccc 480 tggtcaccgt ctcctca
497 33 139 PRT Homo sapiens 33 Met Glu Phe Gly Leu Ser Trp Leu Phe
Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Arg Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu
Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Arg Tyr Tyr Ala 65 70
75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Glu Ser Ser Gly Trp Phe
Gly Ala Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 130 135 34 446 DNA Homo sapiens 34 gatcttaaaa gaggttcttt
ctctgggatg tggcatgagc aaaactgaca agtcaaggca 60 ggaagatgtc
gccatcacaa ctcattgggt ttctgctgct ctgggttcca gcctccaggg 120
gtgaaattgt gctgactcag tctccagact ttcagtctgt gactccaaag gagaaagtca
180 ccatcacctg ccgggccagt cagagcattg gtagtagctt acactggtac
cagcagaaac 240 cagatcagtc tccaaagctc ctcatcaagt atgcttccca
gtccttctca ggggtcccct 300 cgaggttcag tggcagtgga tctgggacag
atttcaccct caccatcaat agcctggaag 360 ctgaagatgc tgcagcgtat
tactgtcatc agagtagtag tttaccgatc accttcggcc 420 aagggacacg
actggagatt aaacga 446 35 127 PRT Homo sapiens 35 Met Ser Pro Ser
Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro
Ala 1 5 10 15 Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Asp Phe
Gln Ser Val 20 25 30 Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile 35 40 45 Gly Ser Ser Leu His Trp Tyr Gln Gln
Lys Pro Asp Gln Ser Pro Lys 50 55 60 Leu Leu Ile Lys Tyr Ala Ser
Gln Ser Phe Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser 85 90 95 Leu Glu Ala
Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser 100 105 110 Leu
Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 115 120 125
36 31 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 36 tcttgtccac cttggtgttg ctgggcttgt g 31 37
30 DNA Artificial Sequence Description of Artificial
SequenceSynthetic DNA 37 aggcacacaa cagaggcagt tccagatttc 30 38 19
DNA Artificial Sequence Description of Artificial SequenceSynthetic
DNA 38 gatttaggtg acactatag 19 39 20 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 39 taatacgact
cactataggg 20 40 41 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 40 atcacagatc tctcaccatg
gaagccccag ctcagcttct c 41 41 33 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 41 ggtgcagcca
ccgtacgttt gatctccacc ttg 33 42 38 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 42 gcgactaagt
cgacaccatg gactggacct ggaggatc 38 43 32 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 43 agagagagag
gctagctgag gagacggtga cc 32 44 27 DNA Artificial Sequence
Description of Artificial SequenceSynthetic DNA 44 ggtacgtgaa
ccgtcagatc gcctgga 27 45 27 DNA Artificial Sequence Description of
Artificial SequenceSynthetic DNA 45 tctatataag cagagctggg tacgtcc
27
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